Production of heteromultimeric proteins

ABSTRACT

Described herein are methods for the efficient production of antibodies and other multimeric protein complexes (collectively referred to herein as heteromultimeric proteins) capable of specifically binding to more than one target. The targets may be, for example, different epitopes on a single molecule or located on different molecules. The methods combine efficient, high gene expression level, appropriate assembly, and ease of purification for the heteromultimeric proteins. The invention also provides methods of using these heteromultimeric proteins, and compositions, kits and articles of manufacture comprising these antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/045,539, filed Jul. 25, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/796,530, filed Oct. 27, 2017 (now abandoned),which is a continuation of U.S. patent application Ser. No. 15/454,968,filed Mar. 9, 2017 (now abandoned), which is a divisional of U.S. patentapplication Ser. No. 13/092,708, filed Apr. 22, 2011, which issued asU.S. Pat. No. 9,637,557 on May 2, 2017, which claims priority to U.S.Provisional patent Application No. 61/327,302, filed Apr. 23, 2010, thedisclosures of all which are hereby incorporated by reference in theirentireties for all purposes.

TECHNICAL FIELD

This invention relates to methods for the production of heteromultimericproteins.

BACKGROUND

Monoclonal antibodies of the IgG type contain two identicalantigen-binding arms and a constant domain (Fc). Antibodies with adiffering specificity in their binding arms usually do not occur innature and, therefore, have to be crafted with the help of chemicalengineering (e.g., chemical cross-linking, etc), recombinant DNA and/orcell-fusion technology.

Bispecific antibodies can bind simultaneously two different antigens.This property enables the development of therapeutic strategies that arenot possible with conventional monoclonal antibodies. The large panel ofimaginative bispecific antibody formats that has been developed reflectsthe strong interest for these molecules. See Berg J, Lotscher E, SteimerK S, et al., “Bispecific antibodies that mediate killing of cellsinfected with human immunodeficiency virus of any strain,” Proc NatlAcad Sci USA (1991) 88(11): 4723-4727 and Fischer N and Leger O.,“Biospecific Antibodies: Molecules That Enable Novel TherapeuticStrategies,” Pathobiology (2007) 74:3-14.

Another class of multispecific molecules is recombinant fusion proteins.Recombinant fusion proteins consisting of the extracellular domain ofimmunoregulatory proteins and the constant (Fc) domain of immunoglobulin(Ig) represent a growing class of human therapeutics. Immunoadhesinscombine the binding region of a protein sequence, with a desiredspecificity, with the effector domain of an antibody. Immunoadhesinshave two important properties that are significant to their potential astherapeutic agents: the target specificity, and the pharmacokineticstability (half-life in vivo that is comparable to that of antibodies).Immunoadhesins can be used as antagonist to inhibit or block deleteriousinteractions or as agonist to mimic or enhance physiological responses.See Chamow S M, Zhang D Z, Tan X Y, et al., “A humanized, bispecificimmunoadhesin-antibody that retargets CD3+ effectors to killHIV-1-infected cells,” J Hematother 1995; 4(5): 439-446.

Other multispecific molecules have been discussed elsewhere. Examplesinclude but are not limited to: Fisher et al., Pathobiology (2007)74:3-14 (review of various bispecific formats); U.S. Pat. No. 6,660,843,issued Dec. 9, 2003 to Feige et al. (peptibodies); US Pat. Publ. No.2002-004587 published Jan. 10, 2002 (multispecific antibodies); U.S.Pat. No. 7,612,181 issued Nov. 3, 2009 to Wu et al. (Dual VariableDomain format); U.S. Pat. No. 6,534,628, Nord K et al., Prot Eng (1995)8:601-608, Nord K et al., Nat Biotech (1997) 15:772-777, and Grönwall etal., Biotechnol Appl Biochem. (2008) June; 50(Pt 2):97-112 (Affibodies);Martens et al., Clin Cancer Res (2006), 12: 6144-6152 and Jin et al.,Cancer Res (2008) 68(11):4360-4368 (one armed antibodies); Bostrom etal., Science (2009) 323:1610-1614 (Dual Action Fab, aka mixed valencyantibodies). Other formats are known to those skilled in the art.

The manufacturing of clinical grade material remains challenging for themultispecific molecules described above. As noted above, there are manypaths to the production of molecules with mixed binding arms, i.e.,binding arms that are not identical to each other. Each of these methodshas its drawbacks.

Chemical cross-linking is labor intensive as the relevant species mayyet need to be purified from homodimers and other undesired by-products.In addition, the chemical modification steps can alter the integrity ofthe proteins thus leading to poor stability. Thus, this method is ofteninefficient and can lead to loss of antibody activity.

Cell-fusion technology (e.g., hybrid hybridomas) express two heavy andtwo light chains that assemble randomly leading to the generation of 10antibody combinations. The desired heteromultimeric antibodies are onlya small fraction of the antibodies thus produced. Purification of thedesired heteromultimeric proteins dramatically reduces production yieldsand increases manufacturing costs.

Recombinant DNA techniques have been used to generate variousheteromultimeric formats, e.g., single chain Fv, diabodies, etc., thatdo not comprise an Fc domain. A major drawback for this type of antibodymolecule is the lack of the Fc domain and thus the ability of theantibody to trigger an effector function (e.g., complement activation,Fc-receptor binding etc.). Thus, a bispecific antibody comprising afunctional Fc domain is desired.

Recombinant DNA techniques have also been used to generate ‘knob intohole’ bispecific antibodies. See US Patent Application 20030078385(Arathoon et al. —Genentech). One constraint of this strategy is thatthe light chains of the two parent antibodies have to be identical toprevent mispairing and formation of undesired and/or inactive moleculesdue to being expressed in the same cell.

Thus, there remains a need for alternative methods of producingheteromultimeric proteins. The invention described herein provides suchmethods. These and other aspects and advantages of the invention will beapparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

Production of heteromultimeric proteins, e.g., multispecific antibodies,using current techniques has drawbacks including the production of amixture of products, reduced yield and decreased/elimination of effectorfunction among others. Thus, it is desirable to produce heteromultimericproteins efficiently and at high levels.

The production of antibody molecules, by various means, is generallywell understood. U.S. Pat. No. 6,331,415 (Cabilly et al.), for example,describes a method for the recombinant production of immunoglobulinwhere the heavy and light chains are expressed simultaneously from asingle vector or from two separate vectors in a single cell. Wibbenmeyeret al., (1999, Biochim Biophys Acta 1430(2): 191-202) and Lee and Kwak(2003, J. Biotechnology 101:189-198) describe the production ofmonoclonal antibodies from separately produced heavy and light chains,using plasmids expressed in separate cultures of E. coli. Various othertechniques relevant to the production of antibodies are described in,e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988) andWO2006028936. Yet each of these have draw backs such as low yield, useof chemicals

The inventive methods provide for the expression of each component,e.g., one arm of an antibody, of a hinge-containing heteromultimericprotein in a separate host cell and the assembly of the hinge-containingheteromultimeric protein, e.g., a multispecific antibody, without theaddition of a reductant.

This invention provides an easy and efficient production process/methodthat allows for the economical production of heteromultimeric proteins,e.g., multispecific antibodies.

The invention provides efficient and novel methods of producingmultispecific immunoglobulin complexes (e.g., multispecific antibodies)and other multimeric proteins (collectively referred to herein asheteromultimeric proteins) that overcome limitations of traditionalmethods. Heteromultimeric proteins, such as bispecific antibodies, canbe provided as a highly homogeneous heteromultimer polypeptide accordingto methods of the invention. In addition, the methods provided forherein do not rely on the addition of a reductant to achieve theformation of at least one, at least two, at least three, at least fourinterchain disulfide bonds in the heteromultimeric protein.

In a first aspect, the method described herein allows for thepreparation of a heteromultimeric protein comprising a firsthinge-containing polypeptide having a first heterodimerization domainand a second hinge-containing polypeptide having a secondheterodimerization domain, wherein the second heterodimerization domaininteracts with the first heterodimerization domain, and wherein thefirst and second hinge-containing polypeptides are linked by at leastone interchain disulfide bond, the method comprising the steps of:

-   -   (a) culturing a first host cell comprising a first nucleic acid        encoding the first hinge-containing polypeptide under conditions        where the hinge-containing polypeptide is expressed;    -   (b) culturing a second host cell comprising a nucleic acid        encoding the second hinge-containing polypeptide under        conditions where the hinge-containing polypeptide is expressed;    -   (c) disrupting the cell membranes so that the first and second        hinge-containing polypeptides are released into the        extracellular milieu, wherein the first and second host cells        have been combined together in a single suspension; and    -   (d) recovering the heteromultimeric protein,        wherein said method does not require the addition of a        reductant.

In a second aspect, the method of preparing a heteromultimeric proteincomprising heteromultimeric protein comprising a first hinge-containingpolypeptide having a first heterodimerization domain and a secondhinge-containing polypeptide having a second heterodimerization domain,wherein the second heterodimerization domain interacts with the firstheterodimerization domain, and wherein the first and secondhinge-containing polypeptides are linked by at least one interchaindisulfide bond, the method comprising the steps of:

-   -   (a) providing a purified first hinge-containing polypeptide        having a first heterodimerization domain;    -   (b) providing a purified second hinge-containing polypeptide        having a second heterodimerization domain;    -   (c) combining the first and second hinge-containing        polypeptides;    -   (d) refolding the first hinge-containing polypeptide with the        second hinge-containing polypeptide; and    -   (e) recovering the heteromultimeric protein complex.

In a third aspect, the methods provided for herein are directed to amethod of preparing a heteromultimeric protein comprising incubating afirst pair of immunoglobulin heavy and light chain polypeptides, and asecond pair of immunoglobulin heavy and light chain polypeptides, underconditions permitting multimerization of the first and second pair ofpolypeptides to form a substantially homogeneous population ofantibodies, wherein the conditions do not comprise the addition of areductant; wherein the first pair of polypeptides is capable of bindinga first target; wherein the second pair of polypeptides is capable ofbinding a second target molecule; and wherein Fc polypeptide of thefirst heavy chain polypeptide and Fc polypeptide of the second heavychain polypeptide meet at an interface, and the interface of the secondFc polypeptide comprises a protuberance which is positionable in acavity in the interface of the first Fc polypeptide.

In a fourth aspect, there is a method of generating a combinatoriallibrary of heteromultimeric proteins, said method comprising a firsthinge-containing polypeptide having a first heterodimerization domainand a second hinge-containing polypeptide having a secondheterodimerization domain, wherein the second heterodimerization domaininteracts with the first heterodimerization domain, and wherein thefirst and second hinge-containing polypeptides are linked by at leastone interchain disulfide bond, the method comprising the steps of:

-   -   (a) culturing a first host cell and at least two additional host        cells, wherein        -   a. said first host cell comprises a first nucleic acid            encoding a first heterodimerization domain-containing            polypeptide; and        -   b. said additional host cells comprise a nucleic acid            comprising a second heterodimerization domain-containing            polypeptide,    -   (b) combining the first and at least two additional host cells;    -   (c) treating the cells so that the first and second        heterodimerization domain-containing polypeptides are released        into the extracellular milieu; and    -   (d) recovering the heteromultimeric proteins, wherein said        method does not require the addition of a reductant.

In a fifth aspect, there are provided the heteromultimeric proteinsproduced by the methods described herein.

It is to be understood that methods of the invention can include othersteps which generally are routine steps evident for initiating and/orcompleting the process encompassed by methods of the invention asdescribed herein. For example, in one embodiment, step (a) of a methodof the invention is preceded by a step wherein a nucleic acid encoding afirst hinge-containing polypeptide is introduced into a first host cell,and a nucleic acid encoding a second hinge-containing polypeptide isintroduced into a second host cell. In one embodiment, methods of theinvention further comprise a step of purifying heteromultimeric proteinshaving binding specificity to at least two distinct targets. In oneembodiment, no more than about 10%, 15%, or 20% of isolated polypeptidesare present as monomers or heavy-light chain dimers prior to the step ofpurifying the heteromultimeric proteins.

In an embodiment, the first and/or second hinge-containing polypeptideis an antibody heavy chain. In a further embodiment, the antibody heavychain is paired with an antibody light chain to provide a heavy-lightchain pair. In some embodiments, the heavy-light chain pair arecovalently linked. In another embodiment, the heavy-light chain pairdefines a target binding arm. In some embodiments, the target bindingarms are identical. In some embodiments, the target binding arms eachrecognize two distinct targets.

In some embodiments, the first and/or second hinge-containingpolypeptide comprises an Fc region. In another embodiment the firstand/or second hinge-containing polypeptide comprises at least oneconstant heavy domain. In another embodiment, the first and/or secondhinge-containing polypeptide comprises a variable heavy chain domain. Inanother embodiment, the first and/or second hinge-containing polypeptidecomprises a receptor binding domain. In some embodiments, the firstand/or second hinge-containing polypeptide are substantially identical(i.e., the heterodimerization domain may not be identical with theregions outside of the heterodimerization domain being identical). Insome embodiments, the first and/or second hinge-containing polypeptideare not identical.

In some embodiments, the heteromultimeric protein is selected from thegroup consisting of an antibody, a bispecific antibody, a multispecificantibody, one-armed antibody, monospecific monovalent antibody, amultispecific monovalent antibody, a bispecific maxibody, a monobody, animmunoadhesin, a peptibody, a bispecific peptibody, a monovalentpeptibody, an affibody and a receptor fusion protein.

In some embodiments, said heteromultimeric proteins comprise a hingeregion that has at least one, at least two, at least three, at leastfour, or any integer number up to all, of the cysteine residues that arenormally capable of forming an inter-heavy chain disulfide linkage. Insome embodiments, additional cysteines have been introduced into thehinge region.

A heteromultimeric protein of the invention may also be an antibodyfragment, such as, for example, an Fc or Fc fusion polypeptide, so longas it comprises the hinge region of an immunoglobulin. An Fc fusionpolypeptide generally comprises an Fc polypeptide (or fragment thereof)fused to a heterologous polypeptide sequence (such as an antigen bindingdomain), such as a receptor extracellular domain (ECD) fused to animmunoglobulin Fc polypeptide (e.g., Flt receptor ECD fused to a IgG2Fc). For example, in one embodiment, an Fc fusion polypeptide comprisesa VEGF binding domain, which may be a VEGF receptor, which includes flt,flk, etc. A heteromultimeric protein of the invention generallycomprises a heavy chain constant domain and a light chain constantdomain. In one embodiment, a heteromultimeric protein of the inventioncomprises a modification (for example, but not limited to, insertion ofone or more amino acids, e.g., to form a dimerization sequence such asleucine zipper) for formation of inter-heavy chain dimerization ormultimerization. In some embodiments, a portion (but not all) of the Fcpolypeptide is missing in a heteromultimer of the invention, so long asit retains the hinge region of an immunoglobulin. In some of theseembodiments, the missing sequence of the Fc polypeptide is a portion orall of the C_(H)2 and/or C_(H)3 domain. In some of these embodiments,the heteromultimeric protein comprises a dimerization domain (such as aleucine zipper sequence), for example fused to the C-terminus of theheavy chain fragment. In some of these embodiments, the heteromultimericprotein comprises a dimerization domain comprising mutations to providefor a “knob into hole” dimerization domain (as further defined below).

In some embodiments of the methods and heteromultimeric proteins of theinvention, the hinge-containing polypeptides comprise at least onecharacteristic that promotes heterodimerization, while minimizinghomodimerization, of the first and second hinge-containing polypeptides(e.g., between Fc polypeptides of the heavy chains). Suchcharacteristic(s) improves yield and/or purity and/or homogeneity of theheteromultimeric protein populations obtainable by methods of theinvention as described herein. In one embodiment, the Fc polypeptides ofa first hinge-containing polypeptide and a second hinge-containingpolypeptide meet/interact at an interface. In some embodiments whereinthe Fc polypeptides of the first and second hinge-containingpolypeptides meet at an interface, the interface of the second Fcpolypeptide comprises a protuberance which is positionable in a cavityin the interface of the first Fc polypeptide. In one embodiment, thefirst Fc polypeptide has been altered from a template/originalpolypeptide to encode the cavity or the second Fc polypeptide has beenaltered from a template/original polypeptide to encode the protuberance,or both. In one embodiment, the first Fc polypeptide has been alteredfrom a template/original polypeptide to encode the cavity and the secondFc polypeptide has been altered from a template/original polypeptide toencode the protuberance, or both. In one embodiment, the interface ofthe second Fc polypeptide comprises a protuberance which is positionablein a cavity in the interface of the first Fc polypeptide, wherein thecavity or protuberance, or both, have been introduced into the interfaceof the first and second Fc polypeptides, respectively. In someembodiments wherein the first and second Fc polypeptides meet at aninterface, the interface of the first Fc polypeptide comprises aprotuberance which is positionable in a cavity in the interface of thesecond Fc polypeptide. In one embodiment, the second Fc polypeptide hasbeen altered from a template/original polypeptide to encode the cavityor the first Fc polypeptide has been altered from a template/originalpolypeptide to encode the protuberance, or both. In one embodiment, thesecond Fc polypeptide has been altered from a template/originalpolypeptide to encode the cavity and the first Fc polypeptide has beenaltered from a template/original polypeptide to encode the protuberance,or both. In one embodiment, the interface of the first Fc polypeptidecomprises a protuberance which is positionable in a cavity in theinterface of the second Fc polypeptide, wherein the protuberance orcavity, or both, have been introduced into the interface of the firstand second Fc polypeptides, respectively.

In one embodiment, the protuberance and cavity each comprises anaturally occurring amino acid residue. In one embodiment, the Fcpolypeptide comprising the protuberance is generated by replacing anoriginal residue from the interface of a template/original polypeptidewith an import residue having a larger side chain volume than theoriginal residue. In one embodiment, the Fc polypeptide comprising theprotuberance is generated by a method comprising a step wherein nucleicacid encoding an original residue from the interface of said polypeptideis replaced with nucleic acid encoding an import residue having a largerside chain volume than the original. In one embodiment, the originalresidue is threonine. In one embodiment, the import residue is arginine(R). In one embodiment, the import residue is phenylalanine (F). In oneembodiment, the import residue is tyrosine (Y). In one embodiment, theimport residue is tryptophan (W). In one embodiment, the import residueis R, F, Y or W. In one embodiment, a protuberance is generated byreplacing two or more residues in a template/original polypeptide. Inone embodiment, the Fc polypeptide comprising a protuberance comprisesreplacement of threonine at position 366 with tryptophan, amino acidnumbering according to the EU numbering scheme of Kabat et al. (pp.688-696 in Sequences of proteins of immunological interest, 5th ed.,Vol. 1 (1991; NIH, Bethesda, Md.)).

In some embodiments, the Fc polypeptide comprising a cavity is generatedby replacing an original residue in the interface of a template/originalpolypeptide with an import residue having a smaller side chain volumethan the original residue. For example, the Fc polypeptide comprisingthe cavity may be generated by a method comprising a step whereinnucleic acid encoding an original residue from the interface of saidpolypeptide is replaced with nucleic acid encoding an import residuehaving a smaller side chain volume than the original. In one embodiment,the original residue is threonine. In one embodiment, the originalresidue is leucine. In one embodiment, the original residue is tyrosine.In one embodiment, the import residue is not cysteine (C). In oneembodiment, the import residue is alanine (A). In one embodiment, theimport residue is serine (S). In one embodiment, the import residue isthreonine (T). In one embodiment, the import residue is valine (V). Acavity can be generated by replacing one or more original residues of atemplate/original polypeptide. For example, in one embodiment, the Fcpolypeptide comprising a cavity comprises replacement of two or moreoriginal amino acids selected from the group consisting of threonine,leucine and tyrosine. In one embodiment, the Fc polypeptide comprising acavity comprises two or more import residues selected from the groupconsisting of alanine, serine, threonine and valine. In someembodiments, the Fc polypeptide comprising a cavity comprisesreplacement of two or more original amino acids selected from the groupconsisting of threonine, leucine and tyrosine, and wherein said originalamino acids are replaced with import residues selected from the groupconsisting of alanine, serine, threonine and valine. In one embodiment,the Fc polypeptide comprising a cavity comprises replacement ofthreonine at position 366 with serine, amino acid numbering according tothe EU numbering scheme of Kabat et al., supra. In one embodiment, theFc polypeptide comprising a cavity comprises replacement of leucine atposition 368 with alanine, amino acid numbering according to the EUnumbering scheme of Kabat et al., supra. In one embodiment, the Fcpolypeptide comprising a cavity comprises replacement of tyrosine atposition 407 with valine, amino acid numbering according to the EUnumbering scheme of Kabat et al., supra. In one embodiment, the Fcpolypeptide comprising a cavity comprises two or more amino acidreplacements selected from the group consisting of T366S, L368A andY407V, amino acid numbering according to the EU numbering scheme ofKabat et al., supra. In some embodiments of these antibody fragments,the Fc polypeptide comprising the protuberance comprises replacement ofthreonine at position 366 with tryptophan, amino acid numberingaccording to the EU numbering scheme of Kabat et al., supra.

In various embodiments, the Fc polypeptide of the first and second heavychain polypeptides may or may not be identical, provided they arecapable of dimerizing to form an Fc region (as defined herein). A firstFc polypeptide is generally contiguously linked to one or more domainsof an immunoglobulin heavy chain in a single polypeptide, for examplewith hinge, constant and/or variable domain sequences. In oneembodiment, the first Fc polypeptide comprises at least a portion(including all) of a hinge sequence, at least a portion (including all)of a C_(H)2 domain and/or at least a portion (including all) of a C_(H)3domain. In one embodiment, the first Fc polypeptide comprises the hingesequence and the C_(H)2 and C_(H)3 domains of an immunoglobulin. In oneembodiment, the second Fc polypeptide comprises at least a portion(including all) of a hinge sequence, at least a portion (including all)of a C_(H)2 domain and/or at least a portion (including all) of a C_(H)3domain. In one embodiment, the second Fc polypeptide comprises the hingesequence and the C_(H)2 and C_(H)3 domains of an immunoglobulin. In oneembodiment, an antibody of the invention comprises first and second Fcpolypeptides each of which comprising at least a portion of at least oneantibody constant domain. In one embodiment, the antibody constantdomain is a C_(H)2 and/or C_(H)3 domain. In any of the embodiments of anantibody of the invention that comprises a constant domain, the antibodyconstant domain can be from any immunoglobulin class, for example anIgG. The immunoglobulin source can be of any suitable species of origin(e.g., an IgG may be human IgG₁) or of synthetic form.

In one embodiment, a first light chain polypeptide and a second lightchain polypeptide in a first and second target molecule binding arm,respectively, of an antibody of the invention comprisedifferent/distinct antigen binding determinants (e.g.,different/distinct variable domain sequences). In one embodiment, afirst light chain polypeptide and a second light chain polypeptide in afirst and second target molecule binding arm, respectively, of anantibody of the invention comprise the same (i.e., a common) antigenbinding determinant e.g., the same variable domain sequence).

Methods of the invention are capable of generating heteromultimericmolecules at high homogeneity. According, the invention provides methodswherein at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% of polypeptides are in a complex comprisinga first heavy and light chain polypeptide pair and a second heavy andlight chain polypeptide pair. In one embodiment, the invention providesmethods wherein between about 60 and 99%, 70 and 98%, 75 and 97%, 80 and96%, 85 and 96%, or 90 and 95% of polypeptides are in a complexcomprising a first heavy and light chain polypeptide pair and a secondheavy and light chain polypeptide pair.

In one embodiment, an antibody of the invention is selected from thegroup consisting of IgG, IgE, IgA, IgM and IgD. In some embodiments, thehinge region of an antibody of the invention is preferably of animmunoglobulin selected from the group consisting of IgG, IgA and IgD.For example, in some embodiments, an antibody or hinge region of anantibody is of IgG, which in some embodiments is IgG1 or IgG2 (e.g.,IgG2a or IgG2b). In some embodiments, an antibody of the invention isselected from the group consisting of IgG, IgA and IgD. In oneembodiment, the antibody is of human, humanized, chimeric or non-human(e.g., murine) origin.

Heteromultimeric proteins of the invention generally are capable ofbinding, preferably specifically, to antigens. Such antigens include,for example, tumor antigens, cell survival regulatory factors, cellproliferation regulatory factors, molecules associated with (e.g., knownor suspected to contribute functionally to) tissue development ordifferentiation, cell surface molecules, lymphokines, cytokines,molecules involved in cell cycle regulation, molecules involved invasculogenesis and molecules associated with (e.g., known or suspectedto contribute functionally to) angiogenesis. An antigen to which aheteromultimeric protein of the invention is capable of binding may be amember of a subset of one of the above-mentioned categories, wherein theother subset(s) of said category comprise other molecules/antigens thathave a distinct characteristic (with respect to the antigen ofinterest). An antigen of interest may also be deemed to belong to two ormore categories. In one embodiment, the invention provides aheteromultimeric protein that binds, preferably specifically, a tumorantigen that is not a cell surface molecule. In one embodiment, a tumorantigen is a cell surface molecule, such as a receptor polypeptide. Inanother example, in some embodiments, a heteromultimeric protein of theinvention binds, preferably specifically, a tumor antigen that is not acluster differentiation factor. In another example, a heteromultimericprotein of the invention is capable of binding, preferably specifically,to a cluster differentiation factor, which in some embodiments is not,for example, CD3 or CD4. In some embodiments, a heteromultimeric proteinof the invention is an anti-VEGF antibody. In some embodiments, aheteromultimeric protein of the invention is a bispecific antibodyselected from the group consisting of IL-1alpha/IL-1beta, IL-12/IL-18;IL-13/IL-9; IL-13/IL-4, IL-13/IL-5, IL-5/IL-4, IL-13/IL-1beta,IL-13/IL-25; IL-13/TARC, IL-13/MDC, IL-13/MEF, IL-13/TGF-β, IL-13/LHRagonist; IL-12/TWEAK, IL-13/CL25, IL-13/SPRR2a, IL-13/SPRR2b;IL-13/ADAM8, IL-13/PED2, IL17A/IL17F, CD3/CD19, CD138/CD20, CD138/CD40,CD19/CD20, CD20/CD3, CD38/CD138, CD38/CD20, CD38/CD40, CD40/CD20,CD-8/IL-6; CD20/BR3, TNFalpha/TGF-beta, TNFalpha/IL-1beta,TNFalpha/IL-2, TNF alpha/IL-3, TNFalpha/IL-4, TNFalpha/IL-5,TNFalpha/IL6, TNFalpha/I L8, TNFalpha/IL-9, TNFalpha/IL-10,TNFalpha/IL-11, TNFalpha/IL-12, TNFalpha/IL-13, TNFalpha/IL-14,TNFalpha/IL-15, TNFalpha/IL-16, TNFalpha/IL-17, TNFalpha/IL-18,TNFalpha/IL-19, TNFalpha/IL-20, TNFalpha/IL-23, TNFalpha/IFNalpha,TNFalpha/CD4, TNFalphaNEGF, TNFalpha/MIF, TNFalpha/ICAM-1,TNFalpha/PGE4, TNFalpha/PEG2, TNFalpha/RANK ligand. TNFalpha/Te38;TNFalpha/BAFF; TNFalpha/CD22, TNFalpha/CTLA-4; TNFalpha/GP130;TNFα/IL-12p40; VEGF/HER2, VEGF-A/HER2, VEGF-A/PDGF, HER1/HER2,VEGF-A/VEGF-C, VEGF-C/VEGF-D, HER2/DR5, VEGF/IL-8, VEGF/MET, VEGFR/METreceptor, VEGFR/EGFR, HER2/CD64, HER2/CD3, HER2/CD16, HER2/HER3;EGFR/HER2, EGFR/HER3, EGFR/HER4, IL-13/CD40L, IL4/CD40L, TNFR1/IL-1R,TNFR1/IL-6R, TNFR1/IL-18R, EpCAM/CD3, MAPG/CD28, EGFR/CD64, CSPGs/RGM A;CTLA-4/BTNO2; IGF1/IGF2, IGF1/2/Erb213, MAG/RGM A; NgR/RGM A; NogoA/RGMA; OMGp/RGM A; PDL-I/CTLA-4, and RGM A/RGM B, IL1β/IL18, NRP1/VEGFA,VEGFA/NRP2, cMET/EGFR, ALK1/BMP9, VEGFA/α5β1, HER1/HER3-BU, and CMV. Insome embodiments, a heteromultimeric protein of the invention binds toat least two target molecules selected from the group consisting of:α5β1, ALK1, BMP9, IL-1alpha, IL-1beta, TARC, MDC, MEF, TGF-β, LHRagonist, TWEAK, CL25, SPRR2a, SPRR2b, ADAMS, PED2, CD3, CD4, CD16, CD19,CD20, CD22, CD28, CD40, CD38, CD64, CD138, CD-8, BR3, TNFalpha,TGF-beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-17A, IL-17F, IL-18, IL-19, IL-20,IL-23, IL-25, IFNalpha, MIF, ICAM-1, PGE4, PEG2, RANK ligand, Te38,BAFF, CTLA-4, GP130, IL-12p40, VEGF, VEGF-A, PDGF, HER1, HER2, HER3,HER3-BU, HER4, VEGF-C, VEGF-D, DR5, cMET, MET, MET receptor, VEGFR,EGFR, CD40L, TNFR1, IL-1R, IL-6R, IL-18R, EpCAM, MAPG, CSPGs, BTNO2,IGF1, IGF2, IGF1/2, Erb2B, MAG, NgR, NogoA, NRP1, NRP2, OMGp, PDL-I, RGMA and RGM B. In some embodiments, a heteromultimeric protein of thisinvention binds to CD3 and at least one additional target moleculeselected from BLR1, BR3, CD19, CD20, CD22, CD72, CD79A, CD79B, CD180(RP105), CR2, FcRH1, FcRH2, FcRH5, FCER2, FCRL4, HLA-DOB, and NAG14.

First and second host cells in methods of the invention can be culturedin any setting that permits expression and isolation of the polypeptidesof interest. For example, in one embodiment, the first host cell and thesecond host cell in a method of the invention are grown as separate cellcultures. In another embodiment, the first host cell and the second hostcell in a method of the invention are grown as a mixed culturecomprising both host cells.

In some embodiments, at least one, at least two, at least three or moreadditional hinge-containing polypeptide expressing host cells may begrown either in the same or separate cultures as the first and/or secondhinge-containing host cells. In some embodiments, the additionalhinge-containing polypeptide(s) comprises the same heterodimerizationdomain as the first hinge-containing polypeptide. In some embodiments,the additional hinge-containing polypeptide(s) comprises the sameheterodimerization domain as the second hinge-containing polypeptide.

Heteromultimeric proteins may be modified to enhance and/or addadditional desired characteristics. Such characteristics includebiological functions such as immune effector functions, a desirable invivo half life/clearance, bioavailability, biodistribution or otherpharmacokinetic characteristics. Such modifications are well known inthe art and can also be determined empirically, and may includemodifications by moieties that may or may not be peptide-based. Forexample, antibodies may be glycosylated or aglycosylated, generallydepending at least in part on the nature of the host cell. Preferably,antibodies of the invention are aglycosylated. An aglycosylated antibodyproduced by a method of the invention can subsequently be glycosylatedby, for example, using in vitro glycosylation methods well known in theart. As described above and herein, heteromultimeric proteins of theinvention can be produced in a prokaryotic cell, such as, for example,E. coli. E. coli-produced heteromultimeric proteins are generallyaglycosylated and lack the biological functions normally associated withglycosylation profiles found in mammalian host cell (e.g., CHO) producedheteromultimeric proteins.

The invention also provides immunoconjugates comprising aheteromultimeric protein of the invention conjugated with a heterologousmoiety. Any heterologous moiety would be suitable so long as itsconjugation to the antibody does not substantially reduce a desiredfunction and/or characteristic of the antibody. For example, in someembodiments, an immunoconjugate comprises a heterologous moiety which isa cytotoxic agent. In some embodiments, said cytotoxic agent is selectedfrom the group consisting of a radioactive isotope, a chemotherapeuticagent and a toxin. In some embodiments, said toxin is selected from thegroup consisting of calichemicin, maytansine and trichothene. In someembodiments, an immunoconjugate comprises a heterologous moiety which isa detectable marker. In some embodiments, said detectable marker isselected from the group consisting of a radioactive isotope, a member ofa ligand-receptor pair, a member of an enzyme-substrate pair and amember of a fluorescence resonance energy transfer pair.

In one aspect, the invention provides compositions comprising aheteromultimeric protein of the invention and a carrier, which in someembodiments is pharmaceutically acceptable.

In another aspect, the invention provides compositions comprising animmunoconjugate as described herein and a carrier, which in someembodiments is pharmaceutically acceptable.

In one aspect, the invention provides a composition comprising apopulation of multispecific heteromultimeric proteins of the invention.As would be evident to one skilled in the art, generally such acomposition would not be completely (i.e., 100%) homogeneous. However,as described herein, methods of the invention are capable of producing asubstantially homogeneous population of multispecific heteromultimericproteins. For example, the invention provides a composition comprisingheteromultimeric proteins, wherein at least 40%, 45%, 50%, 55%, 60%,65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of saidheteromultimeric proteins are a multispecific antibody (e.g., abispecific antibody, etc.) of the invention as described herein.

In one aspect, the invention provides a cell culture comprising a mix ofa first host cell and a second host cell, wherein the first host cellcomprises nucleic acid encoding a first hinge-containing polypeptide,and the second host cell comprises nucleic acid encoding a secondhinge-containing polypeptide, and wherein the two pairs have differenttarget binding specificities. In one aspect, the invention provides acell culture comprising a mix of a first host cell and a second hostcell, wherein the first host cell expresses a first pair of heavy andlight chain polypeptides, and the second host cell expresses a secondpair of heavy and light chain polypeptides, and wherein the two pairshave different target binding specificities.

In another aspect, the invention provides articles of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises a heteromultimeric protein (e.g., an antibody) ofthe invention. In another aspect, the invention provides articles ofmanufacture comprising a container and a composition contained therein,wherein the composition comprises an immunoconjugate as describedherein. In some embodiments, these articles of manufacture furthercomprise instructions for using said composition.

In yet another aspect, the invention provides polynucleotides encoding aheteromultimeric protein of the invention. In still another aspect, theinvention provides polynucleotides encoding an immunoconjugate asdescribed herein.

In one aspect, the invention provides recombinant vectors for expressinga molecule (e.g., an antibody) of the invention. In another aspect, theinvention provides recombinant vectors for expressing an immunoconjugateof the invention.

Any of a number of host cells can be used in methods of the invention.Such cells are known in the art (some of which are described herein) orcan be determined empirically with respect to suitability for use inmethods of the invention using routine techniques known in the art. Inone embodiment, a host cell is prokaryotic. In some embodiments, a hostcell is a gram-negative bacterial cell. In one embodiment, a host cellis E. coli. In some embodiments, the E. coli is of a strain deficient inlipoprotein (Δlpp). In some embodiments, the genotype of an E. coli hostcell lacks degP and prc genes and harbors a mutant spr gene. In oneembodiment, a host cell is mammalian, for example, a Chinese HamsterOvary (CHO) cell.

In one aspect, the invention provides host cells comprising apolynucleotide or recombinant vector of the invention. In oneembodiment, a host cell is a mammalian cell, for example a ChineseHamster Ovary (CHO) cell. In one embodiment, a host cell is aprokaryotic cell. In some embodiments, a host cell is a gram-negativebacterial cell, which in some embodiments is E. coli. Host cells of theinvention may further comprise a polynucleotide or recombinant vectorencoding a molecule the expression of which in a host cell enhancesyield of a heteromultimeric protein in a method of the invention. Forexample, such molecule can be a chaperone protein. In one embodiment,said molecule is a prokaryotic polypeptide selected from the groupconsisting of DsbA, DsbC, DsbG and FkpA. In some embodiments, saidpolynucleotide or recombinant vector encodes both DsbA and DsbC. In someembodiments, an E. coli host cell is of a strain deficient in endogenousprotease activities. In some embodiments, the genotype of an E. colihost cell is that of an E. coli strain that lacks degP and prc genes andharbors a mutant spr gene. In some embodiments, the genotype of an E.coli host cell is Δlpp.

Heteromultimeric proteins of the invention find a variety of uses in avariety of settings. In one example, a heteromultimeric protein of theinvention is a therapeutic antibody. In another example, aheteromultimeric protein of the invention is an agonist antibody. Inanother example, a heteromultimeric protein of the invention is anantagonistic antibody. A heteromultimeric protein of the invention mayalso be a diagnostic antibody. In yet another example, aheteromultimeric protein of the invention is a blocking antibody. Inanother example, a heteromultimeric protein of the invention is aneutralizing antibody.

In one aspect, the invention provides methods of treating or delaying adisease in a subject, said methods comprising administering aheteromultimeric protein of the invention to said subject. In oneembodiment, the disease is cancer. In another embodiment, the disease isassociated with dysregulation of angiogenesis. In another embodiment,the disease is an immune disorder, such as rheumatoid arthritis, immunethrombocytopenic purpura, systemic lupus erythematosus, etc.

In one aspect, the invention provides use of a heteromultimeric protein(e.g., an antibody) of the invention in the preparation of a medicamentfor the therapeutic and/or prophylactic treatment of a disease, such asa cancer, a tumor, a cell proliferative disorder, an immune (such asautoimmune) disorder and/or an angiogenesis-related disorder.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as a cancer, a tumor, acell proliferative disorder, an immune (such as autoimmune) disorderand/or an angiogenesis-related disorder.

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disease, such as a cancer, a tumor, a cell proliferativedisorder, an immune (such as autoimmune) disorder and/or anangiogenesis-related disorder.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a fully oxidized half-antibody. Not shown are the“knob” or “hole” or other heterodimerization domains. The half-antibodydepicted in this figure is an IgG1 isotype. One skilled in the art willappreciate that the other immunoglobulin isotypes can be envisioned ashalf-antibodies with the corresponding inter- and intra-chain bonds. Inan intact Ab the hinge cysteines will form inter-chain disulfide bonds.

FIG. 1B illustrates a full-length bispecific antibody. Not depicted arethe inter-heavy chain disulfide bonds in the hinge region.

FIG. 2A & FIG. 2B illustrates plasmids encoding the knob and holehalf-antibodies, respectively.

FIG. 3A illustrates the production of heteromultimeric proteins, e.g.,bispecific antibodies, using the common light chain method. The producedBsAb has two different heavy chains with each being paired with a commonlight chain.

FIG. 3B illustrates the production of heteromultimeric proteins, e.g.,bispecific antibodies, using separately engineered and expressedhalf-antibodies. The produced BsAb typically has two different heavychains, each paired with its cognate light chain. In this method eachlight chain is not necessarily the same for each half-antibody.

FIG. 4A is a flow diagram for the production of bispecific antibodiesusing separately engineered and expressed half-antibodies. In thismethod, redox chemistry is used.

FIG. 4B shows a Coomassie stained gel. The two half-antibodies wereanalyzed under reducing and non-reducing conditions by SDS-PAGE. Thepredominant fraction is the 75 kD light chain-heavy chain pair for eachhalf-antibody under non-reducing conditions. Under reducing conditions(e.g., treatment with DTT) each chain is visible as a separate band.

FIG. 4C shows the results of ESI-TOF mass spectrometry of ahalf-antibody with and without 1 mM N-ethylmaleimide (NEM) treatment. Nochange in the mass of the half-antibody is observed upon treatment withNEM indicating that all cysteines are fully oxidized. The oxidized hingecysteines are represented as a cyclic disulfide in the depicted aminoacid sequence. The expected mass for the half-antibody is 72,548Daltons, which is what is observed by mass spectrometry indicating nocovalent adducts.

FIG. 4D shows the carboxymethyl (CM) chromatogram, a photo of a SDS-PAGEgel and the deconvoluted mass for the production of ananti-EGFR/anti-c-met bispecific antibody. The CM chromatography producesa single peak that is subsequently analyzed by SDS-PAGE. The major bandon the gel is the full-length (i.e., intact) bispecific antibody. Aminor band can also be seen at the 75 kD range. The major band wassubsequently analyzed by mass spectrometry and indicated that the onlydetectable intact antibody product was in agreement with theoretical MWof an anti-EGFR/anti-c-met bispecific antibody.

FIG. 5A is a flow diagram for the large scale production of bispecificantibodies using separately engineered and expressed half-antibodies.

FIG. 5B is photograph of a gel showing the purified half-antibodies weremostly the ˜75 kD species under non-reducing conditions. Under reducingconditions (e.g., treatment with DTT) each chain is visible as aseparate band.

FIG. 5C shows the results of the SDS-PAGE analysis of the purifiedbispecific after removal of aggregates indicating that the major speciesis the intact bispecific antibody at 150 kD. Also shown are the samesamples under reducing conditions indicating that all isolated productis either a light or heavy antibody chain.

FIG. 6A is a graph showing the biological activity of the antibodies ina TF-2 cell proliferation assay testing neutralization of the cytokinesIL-4 and IL-13. The graph shows that the bispecific possesses similaractivity as the two mammalian-produced, full-length antibodies addedtogether or separately.

FIG. 6B is a panel of three graphs showing the pharmacokinetic (PK)properties of an anti-IL-4/anti-IL-13 bispecific antibody incynomologous monkey for the wild-type Fc and a mutated Fc as determinedby ELISA. The first graph shows the PK properties at a 2 mg/kg dose forthe wild-type Fc. The middle graph shows the PK properties at a 20 mg/kgdose, also for the wild-type Fc. The final graph shows the PK propertiesat a 20 mg/kg dose for the mutant Fc. The bispecific exhibits theexpected two compartment clearances in the animals tested. Females arerepresented by closed symbols and males are represented by open symbols.In three animals, an anti-therapeutic response was seen as indicated bythe sharp decrease in measured antibody in serum at day 21.

FIG. 7 is a photograph of a polyacrylamide gel. Whole fermentation brothwas mixed prior to lysis at varying ratios. After lysis protein wasextracted and loaded onto the gel under non-reducing conditions.Purified bispecifics formed during this procedure are visible as the topband on the gel.

FIG. 8A is a photograph of two polyacrylamide gels comparing thebispecific antibody production when the cells are cultured separately toa co-culture of the cells expressing the half-antibodies. The intactbispecific forms to a much higher level under co-culture conditions.When half-antibodies are expressed and purified independently thenmixed, the half-antibodies form less than 5% of the intact bispecific.Under co-culture conditions, greater than 40% is an intact bispecific asdetermined by 150 kD/(150 kD+75 kD) using Li-Core proteindeterminations.

FIG. 8B is a schematic of a co-culture experiment varying the cellpopulation of the initial inoculation. The ratios used and the relativeamount of full-length bispecific are shown at the bottom of the figure.

FIG. 8C is a photograph of a gel for three separate 10 literfermentation runs of a 1:1 cell ratio of anti-EGFR and anti-c-Met. Eachrun produced as the main product the full-length bispecific indicatingthe reproducibility of the process.

FIG. 8D is a flow chart of the co-culture process for the production ofheteromultimeric proteins, e.g., bispecific antibodies.

FIG. 8E is a chromatogram of the UV absorbance at 280 nm identified twosignificant peaks at retention times 91.79 and 102.35. Subsequentanalysis by mass spectrometry indicated that the intact bispecificantibody was effectively separated from the excess half-antibody.

FIG. 8F shows the analysis of Peak 91.79 from FIG. 8E by SDS-PAGE andmass spectrometry. Decovolution of mass spectrometry data produced asingle peak at 146,051.89 Daltons, which is in agreement with theexpected mass of the bispecific antibody. Contaminating homodimericspecies were not detected.

FIG. 8G is a comparison of the work flows for independent production andco-culture production of heteromultimeric proteins.

FIG. 9A show three chromatograms. The top chromatogram shows noabsorbance peak during the elution for the sample without EDTA. Themiddle chromatogram shows the sample with EDTA has a distinct elutionpeak from which we recovered approximately 1.5 mg protein. The lowerchromatogram shows the sample treated with EDTA and Mg also showed asimilar elution peak from which we recovered 1.1 mg protein. Recoveredproteins from the EDTA sample, EDTA plus Mg sample, and a pool offractions from the same retention time from the untreated EDTA samplewere analyzed by SDS-PAGE under reducing and non-reducing conditions.

FIG. 9B is a photograph of the SDS-PAGE gel described in FIG. 9A. Thesamples treated with EDTA have produced intact bispecific antibody thathas been released into the culture media.

FIG. 9C-1, FIG. 9C-2 and FIG. 9C-3 show the mass spec chromatograms forthe samples recovered and described in FIG. 9A. The samples with theEDTA showed the expected mass for the bispecific antibody and a mass forthe excess half-antibody.

FIG. 9D is a photograph of a SDS-PAGE gel and mass chromatograms of theindicated bands. Lane is MW markers, Lane 2 is anti-IL-13 independentlyexpressed, Lane 3 is antiI-IL-4 independently expressed and Lane 4 is aco-culture of the two cells. Mass spec analysis of all three samplesshows that the co-culture produces the intact bispecific and an excessof one half-antibody, anti-IL-4. This indicates the anti-IL-13half-antibody is stoichiometrically limiting. When half-antibodies areexpressed and purified independently then mixed, the half-antibodiesform approximately 2% (anti-IL-13) and 3% (anti-IL-4) of the intactbispecific. Under co-culture conditions, approximately 60% is an intactbispecific as determined by 150 kD/(150 kD+75 kD) using Li-Core proteindeterminations.

FIG. 9E-1 and FIG. 9E-2 show two HIC chromatograms for two co-culturesthat had different cell ratios in the initial fermentation inoculum asindicated. A clear difference in the product is observed that reflectsthe initial inoculum ratio. Using this approach it becomes apparent thatthe initial inoculum ratio can be altered to achieve optimum productionof the heteromultimeric protein.

FIG. 9F is a panel of four photographs showing the SDS-PAGE analysisunder reducing and non-reducing conditions of eight different bispecificantibodies produced by the co-culture process described herein Thenon-reducing gels for the anti-CD3/anti-CD19 heteromultimeric proteinsis not shown. Arrows indicate the intact bispecific antibodies.

FIG. 10 is a schematic of a matrix approach to screeningheteromultimeric proteins.

FIG. 11 shows two graphs for in vitro activity of bispecific antibodiesproduced using the methods described herein.

FIG. 12 is a graph showing that the anti-EGFR/anti-c-met bispecificpossesses anti-tumor activity in a KP4 pancreatic xenograft in vivomodel.

FIG. 13 is a graph showing that the anti-EGFR/anti-c-met bispecificpossesses anti-tumor activity in an A431 epidermoid carcinoma xenograftin vivo model.

FIG. 14A shows the HIC of knob pre-assembly. FIG. 14B shows the HIC ofhole pre-assembly. FIG. 14C shows the HIC of bispecific post assembly.FIG. 14D is a gel of each arm pre-assembly.

FIG. 15 shows an electrophoretogram of assembled material indicatingthat 86% of the material is fully oxidized.

FIGS. 16A, 16B, 16C and 16D show a characterization of assembledbispecific. FIG. 16A provides a HIC chromatogram of annealed bispecificindicating that the material is >90.5 percent bispecific. FIG. 16Bprovides a gel of purified material. FIG. 16C provides a massspectronomy deconvolution of final sample.

FIG. 16D provides a table of theoretical masses.

FIG. 17 is a schematic of redox procedure (with heat): a) sample isheated for an hour to allow cyclisation of disulfide bonds, b) thencooled and cysteines are reduced using 2 mM DTT for two hours, and c)then concentrated and cysteines are air oxidized by dialysis at roomtemperature.

FIG. 18 is a schematic of redox procedure (without heat): a) sample ismixed for two hours, b) cysteines are reduced using 2 mM DTT for twohours, and c) then concentrated and cysteines are air oxidized whileEDTA is removed by dialysis at room temperature

FIGS. 19A and 19B show analytics of assembled bispecific. FIG. 19Aprovides a HIC chromatogram using redox procedure with heating step.FIG. 19B provides a HIC chromatogram using redox procedure withoutheating step.

ABBREVIATIONS

ADCC=Antibody-dependent cell-mediated cytotoxicity

API=Anti-pathogen immunoadhesins

BPI=Bactericidal/permeability-increasing protein

C1q=Complement factor 1q

CD=Cluster of Differentiation

CDC=Complement-dependent cytotoxicity

CH1 or C_(H)1=Heavy chain first constant domain

CH2 or C_(H)2=Heavy chain second constant domain

CH3 or C_(H)3=Heavy chain third constant domain

CH4 or C_(H)4=Heavy chain fourth constant domain

CL or C_(L)=Light chain constant domain

CTLA=Cytotoxic T lymphocyte-associated molecule

Fc=Fragment crystallizable

Fc(R=Receptor gamma for the Fc portion of IgG

HIV=Human immunodeficiency virus

ICAM=Intercellular adhesion molecule

BsAb=Bispecific antibody

BsDb=Bispecific diabody

dsFv=Disulfide-stabilized Fv

Fc=Constant fragment of an antibody

Fd=V_(H)+C_(H)1 of an antibody

FcR=Fc receptor

Fv=Variable fragment of an antibody

IgG=Immunoglobulin G

mAb=Monoclonal antibody

PBL=Peripheral blood lymphocyte

scDb=Single-chain diabody

scFv=Single-chain Fv

(scFv)₂=scFv-scFv tandem

Tandab=Tandem diabody

VH or V_(H)=Variable domain of the heavy chain of an antibody

VL or V_(L)=Variable domain of the light chain of an antibody

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

Numeric ranges are inclusive of the numbers defining the range.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

I. Definitions

A “heteromultimer”, “heteromultimeric complex”, or “heteromultimericprotein” refers to a molecule comprising at least a firsthinge-containing polypeptide and a second hinge-containing polypeptide,wherein the second hinge-containing polypeptide differs in amino acidsequence from the first hinge-containing polypeptide by at least oneamino acid residue. The heteromultimer can comprise a “heterodimer”formed by the first and second hinge-containing polypeptides or can formhigher order tertiary structures where polypeptides in addition to thefirst and second hinge-containing polypeptides are present. Thepolypeptides of the heteromultimer may interact with each other by anon-peptidic, covalent bond (e.g., disulfide bond) and/or a non-covalentinteraction (e.g., hydrogen bonds, ionic bonds, van der Waals forces,and/or hydrophobic interactions).

As used herein, “heteromultimerization domain” refers to alterations oradditions to a biological molecule so as to promote heteromultimerformation and hinder homomultimer formation. Any heterodimerizationdomain having a strong preference for forming heterodimers overhomodimers is within the scope of the invention. Illustrative examplesinclude but are not limited to, for example, US Patent Application20030078385 (Arathoon et al.—Genentech; describing knob into holes);WO2007147901 (Kjærgaard et al. —Novo Nordisk: describing ionicinteractions); WO 2009089004 (Kannan et al. —Amgen: describingelectrostatic steering effects); U.S. Provisional patent Application61/243,105 (Christensen et al.—Genentech; describing coiled coils). Seealso, for example, Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584(1992) describing leucine zipper or Pack et al., Bio/Technology 11,1271-1277 (1993) describing the helix-turn-helix motif. The phrase“heteromultimerization domain” and “heterodimerization domain” are usedinterchangeably herein.

The phrase “hinge-containing polypeptide” as used herein refers to apolypeptide that comprises a region corresponding to the hinge region ofan immunoglobulin as understood in the art, e.g., the region between theC_(H)1 and C_(H)2 domains of the heavy chain. The “hinge region,” “hingesequence”, and variations thereof, as used herein, includes the meaningknown in the art, which is illustrated in, for example, Janeway'sImmunobiology, (Garland Science, Taylor & Francis Group, LLC, NY) (7thed., 2008); Bloom et al., Protein Science (1997), 6:407-415; Humphreyset al., J. Immunol. Methods (1997), 209:193-202. See also, for example,Burton, Molec. Immunol. 22:161-206 (1985) and Papadea, C. and I. J.Check (1989) “Human immunoglobulin G and immunoglobulin G subclasses:biochemical, genetic, and clinical aspects.” Crit Rev Clin Lab Sci27(1): 27-58. It will be appreciated by one skilled in the art that thenumber of amino acids as well as the number of cysteine residuesavailable for interchain disulfide bond formation varies between theclasses and isotypes of immunoglobulins. All such hinge regions may bein the hinge-containing polypeptides and are within the scope of theinvention.

The term “antibody” herein is used in the broadest sense and refers toany immunoglobulin (Ig) molecule comprising two heavy chains and twolight chains, and any fragment, mutant, variant or derivation thereof solong as they exhibit the desired biological activity (e.g., epitopebinding activity). Examples of antibodies include monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies) and antibody fragments as described herein. An antibody canbe human, humanized and/or affinity matured.

As a frame of reference, as used herein an antibody will refer to thestructure of an immunoglobulin G (IgG). However, one skilled in the artwould understand/recognize that an antibody of any immunoglobulin classmay be utilized in the inventive method described herein. For clarity,an IgG molecule contains a pair of identical heavy chains (HCs) and apair of identical light chains (LCs). Each LC has one variable domain(V_(L)) and one constant domain (CO, while each HC has one variable(V_(H)) and three constant domains (C_(H)1, C_(H)2, and C_(H)3). TheC_(H)1 and C_(H)2 domains are connected by a hinge region. Thisstructure is well known in the art. Reference is made to FIG. 1B.

As used herein, “half-antibody” refers to one immunoglobulin heavy chainassociated with one immunoglobulin light chain. An exemplaryhalf-antibody is depicted in FIG. 1A. One skilled in the art willreadily appreciate that a half-antibody may also have an antigen bindingdomain consisting of a single variable domain.

The term “maxibody” refers to a fusion protein comprising a scFv fusedto an Fc polypeptide. Reference is made to FIG. 8a of WO 2009089004.Reference is made to FIG. 2 of WO 2009089004 for a bispecific maxibody.

The term “C_(H)2 domain” of a human IgG Fc region usually extends fromabout residues 231 to about 340 of the IgG according to the EU numberingsystem. The C_(H)2 domain is unique in that it is not closely pairedwith another domain. Rather, two N-linked branched carbohydrate chainsare interposed between the two C_(H)2 domains of an intact native IgGmolecule. It has been speculated that the carbohydrate may provide asubstitute for the domain-domain pairing and help stabilize the C_(H)2domain. Burton, Molec. Immunol. 22:161-206 (1985).

The term “C_(H)3 domain” comprises the stretch of residues C-terminal toa C_(H)2 domain in an Fc region (i.e., from about amino acid residue 341to about amino acid residue 447 of an IgG according to the EU numberingsystem).

The term “Fc region”, as used herein, generally refers to a dimercomplex comprising the C-terminal polypeptide sequences of animmunoglobulin heavy chain, wherein a C-terminal polypeptide sequence isthat which is obtainable by papain digestion of an intact antibody. TheFc region may comprise native or variant Fc sequences. Although theboundaries of the Fc sequence of an immunoglobulin heavy chain mightvary, the human IgG heavy chain Fc sequence is usually defined tostretch from an amino acid residue at about position Cys226, or fromabout position Pro230, to the carboxyl terminus of the Fc sequence.Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991. The Fcsequence of an immunoglobulin generally comprises two constant domains,a C_(H)2 domain and a C_(H)3 domain, and optionally comprises a C_(H)4domain. By “Fc polypeptide” herein is meant one of the polypeptides thatmake up an Fc region, e.g., a monomeric Fc. An Fc polypeptide may beobtained from any suitable immunoglobulin, such as IgG₁, IgG₂, IgG₃, orIgG₄ subtypes, IgA, IgE, IgD or IgM. The Fc region comprises thecarboxy-terminal portions of both H chains held together by disulfides.The effector functions of antibodies are determined by sequences in theFc region; this region is also the part recognized by Fc receptors (FcR)found on certain types of cells. In some embodiments, an Fc polypeptidecomprises part or all of a wild type hinge sequence (generally at its Nterminus). In some embodiments, an Fc polypeptide does not comprise afunctional or wild type hinge sequence.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an antibody variable domain) and can be assessed usingvarious assays as disclosed, for example, in definitions herein.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG, Fc region(non-A and A allotypes); native sequence human IgG₂ Fc region; nativesequence human IgG₃ Fc region; and native sequence human IgG₄ Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g., from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95%, at leastabout 96%, at least about 97%, at least about 98% or at least about 99%homology therewith.

“Fc component” as used herein refers to a hinge region, a C_(H)2 domainor a C_(H)3 domain of an Fc region.

In certain embodiments, the hinge-containing polypeptide comprises anIgG Fc region, preferably derived from a wild-type human IgG Fc region.By “wild-type” human IgG Fc it is meant a sequence of amino acids thatoccurs naturally within the human population. Of course, just as the Fcsequence may vary slightly between individuals, one or more alterationsmay be made to a wildtype sequence and still remain within the scope ofthe invention. For example, the Fc region may contain additionalalterations that are not related to the present invention, such as amutation in a glycosylation site or inclusion of an unnatural aminoacid.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (V_(H) and V_(L), respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). (See,e.g., Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., page91 (2007).) A single V_(H) or V_(L) domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a V_(H) or V_(L) domain from anantibody that binds the antigen to screen a library of complementaryV_(L) or V_(H) domains, respectively. See, e.g., Portolano et al., J.Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “Fab” as used herein refers to an antigen-binding fragment ofan antibody. As noted above, papain may be used to digest an intactantibody. Papain digestion of antibodies produces two identicalantigen-binding fragments, i.e., “Fab” fragments, and a residual “Fc”fragment (i.e., the Fc region, supra). The Fab fragment consists of anentire L chain along with the variable region domain of the H chain(V_(H)), and the first constant domain of one heavy chain (C_(H)1).

The phrase “antigen binding arm”, “target molecule binding arm”, “targetbinding arm” and variations thereof, as used herein, refers to acomponent part of a heteromultimeric protein of the invention that hasan ability to specifically bind a target of interest. Generally andpreferably, the antigen binding arm is a complex of immunoglobulinpolypeptide sequences, e.g., CDR and/or variable domain sequences of animmunoglobulin light and heavy chain.

A “target” or “target molecule” refers to a moiety recognized by abinding arm of the heteromultimeric protein. For example, if theheteromultimeric protein is an antibody, then the target may be epitopeson a single molecule or on different molecules, or a pathogen or a tumorcell, depending on the context. Similarly, if the heteromultimericprotein is a receptor-Fc fusion protein the target would be the cognatebinding partner for the receptor. One skilled in the art will appreciatethat the target is determined by the binding specificity of the targetbinding arm and that different target binding arms may recognizedifferent targets. A target preferably binds to a heteromultimericprotein of this invention with affinity higher than 1 uM Kd (accordingto scatchard analysis). Examples of target molecules include, but arenot limited to, serum soluble proteins and/or their receptors, such ascytokines and/or cytokine receptors, adhesins, growth factors and/ortheir receptors, hormones, viral particles (e.g., RSV F protein, CMV,StaphA, influenza, hepatitis C virus), micoorganisms (e.g., bacterialcell proteins, fungal cells), adhesins, CD proteins and their receptors.

One example of an “intact” or “full-length” antibody is one thatcomprises an antigen-binding arm as well as a C_(L) and at least heavychain constant domains, C_(H)1, C_(H)2, and C_(H)3. The constant domainscan be native sequence constant domains (e.g., human native sequenceconstant domains) or amino acid sequence variants thereof.

The term “coupling” as used herein refers to the steps necessary to linkthe first and second hinge-containing polypeptides to each other, e.g.,formation of a covalent bond. Such steps comprise the reducing,annealing and/or oxidizing of cysteine residues in the first and secondhinge-containing polypeptides to form an inter-chain disulfide bond. Thecoupling may be achieved by chemical cross-linking or the use of a redoxsystem. See, e.g., Humphreys et al., J. Immunol. Methods (1998) 217:1-10and Zhu et al., Cancer Lett., (1994) 86: 127-134.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody that has polyepitopic specificity. Suchmultispecific antibodies include, but are not limited to, an antibodycomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), wherein the V_(H)V_(L) unit has polyepitopicspecificity, antibodies having two or more V_(L) and V_(H) domains witheach V_(H)V_(L) unit binding to a different epitope, antibodies havingtwo or more single variable domains with each single variable domainbinding to a different epitope, full length antibodies, antibodyfragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodiesand triabodies, antibody fragments that have been linked covalently ornon-covalently. “Polyepitopic specificity” refers to the ability tospecifically bind to two or more different epitopes on the same ordifferent target(s). “Monospecific” refers to the ability to bind onlyone epitope. According to one embodiment the multispecific antibody isan IgG antibody that binds to each epitope with an affinity of 5 μM to0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1μM to 0.001 pM. An illustrative drawing of a bispecific is provided inFIG. 1B.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or a variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies (Db); tandem diabodies (taDb), linear antibodies(e.g., U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng.8(10):1057-1062 (1995)); one-armed antibodies, single variable domainantibodies, minibodies, single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments (e.g., includingbut not limited to, Db-Fc, taDb-Fc, taDb-C_(H)3 and (scFV)4-Fc).

The expression “single domain antibodies” (sdAbs) or “single variabledomain (SVD) antibodies” generally refers to antibodies in which asingle variable domain (V_(H) or V_(L)) can confer antigen binding. Inother words, the single variable domain does not need to interact withanother variable domain in order to recognize the target antigen. Singledomain antibodies consist of a single monomeric variable antibody domain(V_(H) or V_(L)) on each antigen binding arm. Examples of single domainantibodies include those derived from camelids (llamas and camels) andcartilaginous fish (e.g., nurse sharks) and those derived fromrecombinant methods from humans and mouse antibodies (Ward et al.,Nature (1989) 341:544-546; Dooley and Flajnik, Dev Comp Immunol (2006)30:43-56; Muyldermans et al., Trend Biochem Sci (2001) 26:230-235; Holtet al., Trends Biotechnol (2003):21:484-490; WO 2005/035572; WO03/035694; Davies and Riechmann, Febs Lett (1994) 339:285-290;WO00/29004; WO 02/051870). A single variable domain antibody can bepresent in an antigen binding arm (e.g., homo- or hetero-multimer) withother variable regions or variable domains, in which case it is not asingle domain antibody.

The expression “linear antibodies” generally refers to the antibodiesdescribed in Zapata et al., Protein Eng. 8(10):1057-1062 (1995).Briefly, these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The term “knob-into-hole” or “KnH” technology as mentioned herein refersto the technology directing the pairing of two polypeptides together invitro or in vivo by introducing a protuberance (knob) into onepolypeptide and a cavity (hole) into the other polypeptide at aninterface in which they interact. For example, KnHs have been introducedin the Fc:Fc binding interfaces, C_(L):C_(H)1 interfaces or V_(H)/V_(L)interfaces of antibodies (e.g., US2007/0178552, WO 96/027011, WO98/050431 and Zhu et al. (1997) Protein Science 6:781-788). This isespecially useful in driving the pairing of two different heavy chainstogether during the manufacture of multispecific antibodies. Forexample, multispecific antibodies having KnH in their Fc regions canfurther comprise single variable domains linked to each Fc region, orfurther comprise different heavy chain variable domains that pair withsimilar or different light chain variable domains. KnH technology can bealso be used to pair two different receptor extracellular domainstogether or any other polypeptide sequences that comprises differenttarget recognition sequences (e.g., including affibodies, peptibodiesand other Fc fusions).

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeCDRs specific for an antigen) has the ability to recognize and bindantigen, although often at a lower affinity than the entire bindingsite.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains,which enables the sFv to form the desired structure for antigen binding.For a review of sFv, see Pluckthun, The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Malmborg et al., J. Immunol. Methods 183:7-13,1995.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term “one-armed antibody” or “one-armed antibodies” refers to anantibody that comprises (1) a variable domain joined by a peptide bondto polypeptide comprising a C_(H)2 domain, a C_(H)3 domain or aC_(H)2-C_(H)3 domain and (2) a second C_(H)2, C_(H)3 or C_(H)2-C_(H)3domain, wherein a variable domain is not joined by a peptide bond to apolypeptide comprising the second C_(H)2, C_(H)3 or C_(H)2-C_(H)3domain. In one embodiment, the one-armed antibody comprises 3polypeptides (1) a first polypeptide comprising a variable domain (e.g.,V_(H)), C_(H)1, C_(H)2 and C_(H)3, (2) a second polypeptide comprising avariable domain (e.g., V_(L)) and a C_(L) domain, and (3) a thirdpolypeptide comprising a C_(H)2 and C_(H)3 domain. In anotherembodiment, the one-armed antibody has a partial hinge region containingthe two cysteine residues which form disulphide bonds linking theconstant heavy chains. In one embodiment, the variable domains of theone armed antibody form an antigen binding region. In anotherembodiment, the variable domains of the one armed antibody are singlevariable domains, wherein each single variable domain is an antigenbinding region. In an embodiment, the one-armed antibody is a singlevariable domain antibody.

Antibodies of the invention can be “chimeric” antibodies in which aportion of the heavy and/or light chain is identical with or homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, provided that they exhibit the desired biologicalactivity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interestherein include primatized antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g., OldWorld Monkey, Ape, etc.) and human constant region sequences.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies cancomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Peptibody” or “peptibodies” refers to a fusion of randomly generatedpeptides with an Fc domain. See U.S. Pat. No. 6,660,843, issued Dec. 9,2003 to Feige et al. (incorporated by reference in its entirety). Theyinclude one or more peptides linked to the N-terminus, C-terminus, aminoacid sidechains, or to more than one of these sites. Peptibodytechnology enables design of therapeutic agents that incorporatepeptides that target one or more ligands or receptors, tumor-homingpeptides, membrane-transporting peptides, and the like. Peptibodytechnology has proven useful in design of a number of such molecules,including linear and disulfide-constrained peptides, “tandem peptidemultimers” (i.e., more than one peptide on a single chain of an Fcdomain). See, for example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No.2003/0195156, published Oct. 16, 2003 (corresponding to WO 02/092620,published Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, publishedSep. 18, 2003 (corresponding to WO 03/031589, published Apr. 17, 2003);U.S. Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023,published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S.Pat. App. No. 2003/0236193, published Dec. 25, 2003 (corresponding toPCT/US04/010989, filed Apr. 8, 2004); U.S. Ser. No. 10/666,480, filedSep. 18, 2003 (corresponding to WO 04/026329, published Apr. 1, 2004),each of which is hereby incorporated by reference in its entirety.

“Affibodies” or “Affibody” refers to the use of a protein liked bypeptide bond to an Fc region, wherein the protein is used as a scaffoldto provide a binding surface for a target molecule. The protein is oftena naturally occurring protein such as staphylococcal protein A orIgG-binding B domain, or the Z protein derived therefrom (see Nilsson etal (1987), Prot Eng 1, 107-133, and U.S. Pat. No. 5,143,844) or afragment or derivative thereof. For example, affibodies can be createdfrom Z proteins variants having altered binding affinity to targetmolecule(s), wherein a segment of the Z protein has been mutated byrandom mutagenesis to create a library of variants capable of binding atarget molecule. Examples of affibodies include U.S. Pat. No. 6,534,628,Nord Ketal, Prot Eng 8:601-608 (1995) and Nord K et al, Nat Biotech15:772-777 (1997). Biotechnol Appl Biochem. 2008 June; 50(Pt 2):97-112.

As used herein, the term “immunoadhesin” designates molecules whichcombine the binding specificity of a heterologous protein (an “adhesin”)with the effector functions of immunoglobulin constant domains.Structurally, the immunoadhesins comprise a fusion of an amino acidsequence with a desired binding specificity, which amino acid sequenceis other than the antigen recognition and binding site of an antibody(i.e., is “heterologous” compared to a constant region of an antibody),and an immunoglobulin constant domain sequence (e.g., C_(H)2 and/orC_(H)3 sequence of an IgG). Exemplary adhesin sequences includecontiguous amino acid sequences that comprise a portion of a receptor ora ligand that binds to a protein of interest. Adhesin sequences can alsobe sequences that bind a protein of interest, but are not receptor orligand sequences (e.g., adhesin sequences in peptibodies). Suchpolypeptide sequences can be selected or identified by various methods,include phage display techniques and high throughput sorting methods.The immunoglobulin constant domain sequence in the immunoadhesin can beobtained from any immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4subtypes, IgA (including IgA1 and IgA2), IgE, IgD, or IgM.

“Complex” or “complexed” as used herein refers to the association of twoor more molecules that interact with each other through bonds and/orforces (e.g., van der waals, hydrophobic, hydrophilic forces) that arenot peptide bonds. In one embodiment, the complex is heteromultimeric.It should be understood that the term “protein complex” or “polypeptidecomplex” as used herein includes complexes that have a non-proteinentity conjugated to a protein in the protein complex (e.g., including,but not limited to, chemical molecules such as a toxin or a detectionagent).

A heteromultimeric protein of this invention “which binds an antigen ofinterest is one that binds the target with sufficient affinity such thatthe heteromultimeric protein is useful as a diagnostic and/ortherapeutic agent in targeting a protein or a cell or tissue expressingthe target, and does not significantly cross-react with other proteins.In such embodiments, the extent of binding of the heteromultimericprotein to a “non-target” protein will be less than about 10% of thebinding of the antibody to its particular target protein as determinedby fluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA) or ELISA. With regard to the binding of aheteromultimeric protein to a target molecule, the term “specificbinding” or “specifically binds to” or is “specific for” a particularpolypeptide or an epitope on a particular polypeptide target meansbinding that is measurably different from a non-specific interaction(e.g., a non-specific interaction may be binding to bovine serum albuminor casein). Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule. For example, specific binding can be determined by competitionwith a control molecule that is similar to the target, for example, anexcess of non-labeled target. In this case, specific binding isindicated if the binding of the labeled target to a probe iscompetitively inhibited by excess unlabeled target. The term “specificbinding” or “specifically binds to” or is “specific for” a particularpolypeptide or an epitope on a particular polypeptide target as usedherein can be exhibited, for example, by a molecule having a Kd for thetarget of at least about 200 nM, alternatively at least about 150 nM,alternatively at least about 100 nM, alternatively at least about 60 nM,alternatively at least about 50 nM, alternatively at least about 40 nM,alternatively at least about 30 nM, alternatively at least about 20 nM,alternatively at least about 10 nM, alternatively at least about 8 nM,alternatively at least about 6 nM, alternatively at least about 4 nM,alternatively at least about 2 nM, alternatively at least about 1 nM, orgreater. In one embodiment, the term “specific binding” refers tobinding where a heteromultimeric protein binds to a particularpolypeptide or epitope on a particular polypeptide without substantiallybinding to any other polypeptide or polypeptide epitope.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). For example, the Kd can be about 200 nM, 150nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM,2 nM, 1 nM, or stronger. Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention.

In one embodiment, the “Kd” or “Kd value according to this invention ismeasured by using surface plasmon resonance assays using a BIAcore™-2000or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized target (e.g., antigen) CM5 chips at ˜10 response units (RU).Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen et al., J.Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M⁻¹ S⁻¹ by thesurface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

“Biologically active” and “biological activity” and “biologicalcharacteristics” with respect to a heteromultimeric protein of thisinvention, such as an antibody, fragment, or derivative thereof, meanshaving the ability to bind to a biological molecule, except wherespecified otherwise.

“Isolated,” when used to describe the various heteromultimerpolypeptides means a heteromultimer which has been separated and/orrecovered from a cell or cell culture from which it was expressed.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for theheteromultimer, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In certain embodiments, theheteromultimer will be purified (1) to greater than 95% by weight ofprotein as determined by the Lowry method, and most preferably more than99% by weight, (2) to a degree sufficient to obtain at least 15 residuesof N-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Ordinarily, however, isolated polypeptide will be prepared by atleast one purification step.

The heteromultimers of the present invention are generally purified tosubstantial homogeneity. The phrases “substantially homogeneous”,“substantially homogeneous form” and “substantial homogeneity” are usedto indicate that the product is substantially devoid of by-productsoriginated from undesired polypeptide combinations (e.g.,homomultimers).

Expressed in terms of purity, substantial homogeneity means that theamount of by-products does not exceed 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2% or1% by weight or is less than 1% by weight. In one embodiment, theby-product is below 5%.

“Biological molecule” refers to a nucleic acid, a protein, acarbohydrate, a lipid, and combinations thereof. In one embodiment, thebiologic molecule exists in nature.

By “linked” or “links as used herein is meant either a direct peptidebond linkage between a first and second amino acid sequence or a linkagethat involves a third amino acid sequence that is peptide bonded to andbetween the first and second amino acid sequences. For example, a linkerpeptide bonded to the C-terminal end of one amino acid sequence and tothe N-terminal end of the other amino acid sequence.

By “linker” as used herein is meant an amino acid sequence of two ormore amino acids in length. The linker can consist of neutral polar ornonpolar amino acids. A linker can be, for example, 2 to 100 amino acidsin length, such as between 2 and 50 amino acids in length, for example,3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. Alinker can be “cleavable,” for example, by auto-cleavage, or enzymaticor chemical cleavage. Cleavage sites in amino acid sequences and enzymesand chemicals that cleave at such sites are well known in the art andare also described herein.

By a “tether” as used herein is meant an amino acid linker that joinstwo other amino acid sequences. A tether as described herein can linkthe N-terminus of an immunoglobulin heavy chain variable domain with theC-terminus of an immunoglobulin light chain constant domain. Inparticular embodiments, a tether is between about 15 and 50 amino acidsin length, for example, between 20 and 26 amino acids in length (e.g.,20, 21, 22, 23, 24, 25, or 26 amino acids in length). A tether may be“cleavable,” for example, by auto-cleavage, or enzymatic or chemicalcleavage using methods and reagents standard in the art.

Enzymatic cleavage of a “linker” or a “tether” may involve the use of anendopeptidase such as, for example, Lys-C, Asp-N, Arg-C, V8, Glu-C,chymotrypsin, trypsin, pepsin, papain, thrombin, Genenase, Factor Xa,TEV (tobacco etch virus cysteine protease), Enterokinase, HRV C3 (humanrhinovirus C3 protease), Kininogenase, as well as subtilisin-likeproprotein convertases (e.g., Furin (PC1), PC2, or PC3) or N-argininedibasic convertase. Chemical cleavage may involve use of, for example,hydroxylamine, N-chlorosuccinimide, N-bromosuccinimide, or cyanogenbromide.

A “Lys-C endopeptidase cleavage site” as used herein is a Lysine residuein an amino acid sequence that can be cleaved at the C-terminal side byLys-C endopeptidase. Lys-C endopeptidase cleaves at the C-terminal sideof a Lysine residue.

By a “chaotropic agent” is meant a water-soluble substance whichdisrupts the three-dimensional structure of a protein (e.g., anantibody) by interfering with stabilizing intra-molecular interactions(e.g., hydrogen bonds, van der Waals forces, or hydrophobic effects).Exemplary chaotropic agents include, but are not limited to, urea,Guanidine-HCl, lithium perchlorate, Histidine, and Arginine.

By a “mild detergent” is meant a water-soluble substance which disruptsthe three-dimensional structure of a protein (e.g., an antibody) byinterfering with stabilizing intra-molecular interactions (e.g.,hydrogen bonds, van der Waals forces, or hydrophobic effects), but whichdoes not permanently disrupt the protein structure as to cause a loss ofbiological activity (i.e., does not denature the protein). Exemplarymild detergents include, but are not limited to, Tween (e.g., Tween-20),Triton (e.g., Triton X-100), NP-40 (nonyl phenoxylpolyethoxylethanol),Nonidet P-40 (octyl phenoxylpolyethoxylethanol), and Sodium DodecylSulfate (SDS).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC refers to aform of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxic agents. The antibodies “arm” thecytotoxic cells and are absolutely required for such killing. Theprimary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of amolecule of interest, an in vitro ADCC assay, such as that described inU.S. Pat. No. 5,500,362 or 5,821,337 can be performed. Useful effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and Natural Killer (NK) cells. Alternatively, or additionally, ADCCactivity of the molecule of interest can be assessed in vivo, e.g., in aanimal model such as that disclosed in Clynes et al., Proc. Natl. Acad.Sci. USA 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a human FcR. Moreover, a preferredFcR is one that binds an IgG antibody (a gamma receptor) and includesreceptors of the FcγRI, FcγRII, and FcγRII) subclasses, includingallelic variants and alternatively spliced forms of these receptors.FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB(an “inhibiting receptor”), which have similar amino acid sequences thatdiffer primarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain (see review M. Daeron, Annu. Rev. Immunol. 15:203-234(1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol.9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, includingthose to be identified in the future, are encompassed by the term “FcR”herein. The term also includes the neonatal receptor, FcRn, which isresponsible for the transfer of maternal IgGs to the fetus (Guyer etal., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249(1994)).

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocytesthat mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells, andneutrophils; with PBMCs and NK cells being preferred. The effector cellscan be isolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)that are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), can be performed.

The term “therapeutically effective amount” refers to an amount of anantibody, antibody fragment, or derivative to treat a disease ordisorder in a subject. In the case of tumor (e.g., a cancerous tumor),the therapeutically effective amount of the antibody or antibodyfragment (e.g., a multispecific antibody or antibody fragment) mayreduce the number of cancer cells; reduce the primary tumor size;inhibit (i.e., slow to some extent and preferably stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentand preferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the disorder. To the extent the antibody or antibodyfragment may prevent growth and/or kill existing cancer cells, it may becytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can,for example, be measured by assessing the duration of survival, time todisease progression (TTP), the response rates (RR), duration ofresponse, and/or quality of life.

By “reduce or inhibit” is meant the ability to cause an overall decreasepreferably of 20% or greater, more preferably of 50% or greater, andmost preferably of 75%, 85%, 90%, 95%, or greater. Reduce or inhibit canrefer to the symptoms of the disorder being treated, the presence orsize of metastases, the size of the primary tumor, or the size or numberof the blood vessels in angiogenic disorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Included in this definition arebenign and malignant cancers. Examples of cancer include but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer (e.g., renal cellcarcinoma), liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma,and various types of head and neck cancer. By “early stage cancer” ismeant a cancer that is not invasive or metastatic or is classified as aStage 0, I, or II cancer. The term “precancerous” refers to a conditionor a growth that typically precedes or develops into a cancer. By“non-metastatic” is meant a cancer that is benign or that remains at theprimary site and has not penetrated into the lymphatic or blood vesselsystem or to tissues other than the primary site. Generally, anon-metastatic cancer is any cancer that is a Stage 0, I, or II cancer,and occasionally a Stage III cancer.

An “allergic or inflammatory disorder” herein is a disease or disorderthat results from a hyper-activation of the immune system of anindividual. Exemplary allergic or inflammatory disorders include, butare not limited to, asthma, psoriasis, rheumatoid arthritis, atopicdermatitis, multiple sclerosis, systemic lupus, erythematosus, eczema,organ transplantation, age-related mucular degeneration, Crohn'sdisease, ulcerative colitis, eosinophilic esophagitis, and autoimmunediseases associated with inflammation.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis such as acute arthritis, chronicrheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronicinflammatory arthritis, degenerative arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebralarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis,arthritis chronica progrediente, arthritis deformans, polyarthritischronica primaria, reactive arthritis, and ankylosing spondylitis),inflammatory hyperproliferative skin diseases, psoriasis such as plaquepsoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of thenails, dermatitis including contact dermatitis, chronic contactdermatitis, allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, colitis such as ulcerative colitis, colitisulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,necrotizing enterocolitis, and transmural colitis, and autoimmuneinflammatory bowel disease), pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, episcleritis), respiratory distresssyndrome, including adult or acute respiratory distress syndrome (ARDS),meningitis, inflammation of all or part of the uvea, iritis,choroiditis, an autoimmune hematological disorder, rheumatoidspondylitis, sudden hearing loss, IgE-mediated diseases such asanaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and/or brainstem encephalitis,uveitis, such as anterior uveitis, acute anterior uveitis, granulomatousuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterioruveitis, or autoimmune uveitis, glomerulonephritis (GN) with and withoutnephrotic syndrome such as chronic or acute glomerulonephritis such asprimary GN, immune-mediated GN, membranous GN (membranous nephropathy),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, allergic reaction, eczemaincluding allergic or atopic eczema, asthma such as asthma bronchiale,bronchial asthma, and auto-immune asthma, conditions involvinginfiltration of T cells and chronic inflammatory responses, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, systemic lupus erythematosus (SLE) or systemiclupus erythematodes such as cutaneous SLE, subacute cutaneous lupuserythematosus, neonatal lupus syndrome (NLE), lupus erythematosusdisseminatus, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I)diabetes mellitus, including pediatric insulin-dependent diabetesmellitus (IDDM), adult onset diabetes mellitus (Type II diabetes),autoimmune diabetes, idiopathic diabetes insipidus, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includinglymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis,vasculitides, including vasculitis (including large vessel vasculitis(including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), microscopic polyarteritis, CNS vasculitis,necrotizing, cutaneous, or hypersensitivity vasculitis, systemicnecrotizing vasculitis, and ANCA-associated vasculitis, such asChurg-Strauss vasculitis or syndrome (CSS)), temporal arteritis,aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia,Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiaperniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA),Factor VIII deficiency, hemophilia A, autoimmune neutropenia,pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNSinflammatory disorders, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet's orBehcet's disease, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, immune complexnephritis, antibody-mediated nephritis, neuromyelitis optica,polyneuropathies, chronic neuropathy such as IgM polyneuropathies orIgM-mediated neuropathy, thrombocytopenia (as developed by myocardialinfarction patients, for example), including thrombotic thrombocytopenicpurpura (TTP) and autoimmune or immune-mediated thrombocytopenia such asidiopathic thrombocytopenic purpura (ITP) including chronic or acuteITP, autoimmune disease of the testis and ovary including autoimmuneorchitis and oophoritis, primary hypothyroidism, hypoparathyroidism,autoimmune endocrine diseases including thyroiditis such as autoimmunethyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto'sthyroiditis), or subacute thyroiditis, autoimmune thyroid disease,idiopathic hypothyroidism, Grave's disease, polyglandular syndromes suchas autoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis,bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barrésyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, primary biliary cirrhosis,pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AIED), autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal garnmopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal segmental glomerulosclerosis (FSGS),endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases,diabetic nephropathy, Dressler's syndrome, alopecia areata, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis,cystic fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis,Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection,echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirusinfection, rubella virus infection, post-vaccination syndromes,congenital rubella infection, Epstein-Barr virus infection, mumps,Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrineophthamopathy, chronic hypersensitivity pneumonitis,keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathicnephritic syndrome, minimal change nephropathy, benign familial andischemia-reperfusion injury, retinal autoimmunity, joint inflammation,bronchitis, chronic obstructive airway disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antibodies, non-malignant thymoma,vitiligo, SCID and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLeishmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune disorders associated with collagendisease, rheumatism, neurological disease, ischemic re-perfusiondisorder, reduction in blood pressure response, vascular dysfunction,antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,reperfusion injury of myocardial or other tissues, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, acute serious inflammation, chronicintractable inflammation, pyelitis, pneumonocirrhosis, diabeticretinopathy, diabetic large-artery disorder, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of a cell and/or causes destruction ofa cell. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², Ra²²³, F³², andradioactive isotopes of Lu), chemotherapeutic agents, e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor, anticancer, and chemotherapeuticagents disclosed herein. Other cytotoxic agents are described herein. Atumoricidal agent causes destruction of tumor cells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1),eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma 1 (see, e.g., Agnew, Chem Intl. Ed. Engl.33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin;as well as neocarzinostatin chromophore and related chromoproteinenediyne antibiotic chromophores), aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, carabicin,carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH, lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.Thus, the growth inhibitory agent may be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (e.g., vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Theagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Anti-cancer therapy” as used herein refers to a treatment that reducesor inhibits cancer in a subject. Examples of anti-cancer therapy includecytotoxic radiotherapy as well as the administration of atherapeutically effective amount of a cytotoxic agent, achemotherapeutic agent, a growth inhibitory agent, a cancer vaccine, anangiogenesis inhibitor, a prodrug, a cytokine, a cytokine antagonist, acorticosteroid, an immunosuppressive agent, an anti-emetic, an antibodyor antibody fragment, or an analgesic to the subject.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). Prodrugs include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, beta-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use inthis invention include, but are not limited to, those chemotherapeuticagents described above.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone (HGH), N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); epidermal growth factor (EGF); hepatic growthfactor; fibroblast growth factor (FGF); prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrins thrombopoietin (TPO); nerve growthfactors such as NGF-alpha; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and erythropoietin (EPO); osteoinductive factors; interferonssuch as interferon-alpha, -beta and -gamma colony stimulating factors(CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF(GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1,IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12; IL-18 a tumor necrosis factor such as TNF-alpha orTNF-beta, and other polypeptide factors including LIF and kit ligand(KL). As used herein, the term cytokine includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

By “cytokine antagonist” is meant a molecule that partially or fullyblocks, inhibits, or neutralizes a biological activity of at least onecytokine. For example, the cytokine antagonists may inhibit cytokineactivity by inhibiting cytokine expression and/or secretion, or bybinding to a cytokine or to a cytokine receptor. Cytokine antagonistsinclude antibodies, synthetic or native-sequence peptides,immunoadhesins, and small-molecule antagonists that bind to a cytokineor cytokine receptor. The cytokine antagonist is optionally conjugatedwith or fused to a cytotoxic agent. Exemplary TNF antagonists areetanercept (ENBREL®), infliximab (REMICADE®), and adalimumab (HUMIRA™).

The term “immunosuppressive agent” as used herein refers to substancesthat act to suppress or mask the immune system of the subject beingtreated. This includes substances that suppress cytokine production,downregulate or suppress self-antigen expression, or mask the MHCantigens. Examples of immunosuppressive agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);mycophenolate mofetil such as CELLCEPT®, azathioprine ° MURANO,AZASAN®/6-mercaptopurine, bromocryptine; danazol; dapsone;glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat.No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHCfragments; cyclosporin A; steroids such as corticosteroids andglucocorticosteroids, e.g., prednisone, prednisolone such as PEDIAPRED®(prednisolone sodium phosphate) or ORAPRED® (prednisolone sodiumphosphate oral solution), methylprednisolone, and dexamethasone;methotrexate (oral or subcutaneous) (RHEUMATREX®, TREXALL™);hydroxycloroquine/chloroquine; sulfasalazine; leflunomide; cytokine orcytokine receptor antagonists including anti-interferon-γ, -β, or -αantibodies, anti-tumor necrosis factor-α antibodies (infliximab oradalimumab), anti-TNFα immunoadhesin (ENBREL®, etanercept), anti-tumornecrosis factor-6 antibodies, anti-interleukin-2 antibodies andanti-IL-2 receptor antibodies; anti-LFA-1 antibodies, includinganti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologousanti-lymphocyte globulin; polyclonal or pan-T antibodies, or monoclonalanti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3binding domain (WO 90/08187); streptokinase; TGF-β, streptodornase; RNAor DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin;T-cell receptor (Cohen et al., U.S. Pat. No. 5,114,721); T-cell receptorfragments (Offner et al. Science 251: 430-432 (1991); WO 90/11294;laneway, Nature 341:482 (1989); and WO 91/01133); T cell receptorantibodies (EP 340,109) such as T10B9, cyclophosphamide (CYTOXAN®);dapsone; penicillamine (CUPRIMINE®); plasma exchange; or intravenousimmunoglobulin (IVIG). These may be used alone or in combination witheach other, particularly combinations of steroid and anotherimmunosuppressive agent or such combinations followed by a maintenancedose with a non-steroid agent to reduce the need for steroids.

An “analgesic” refers to a drug that acts to inhibit or suppress pain ina subject. Exemplary analgesics include non-steroidal anti-inflammatorydrugs (NSAIDs) including ibuprofen (MOTRIN®), naproxen (NAPROSYN®),acetylsalicylic acid, indomethacin, sulindac, and tolmetin, includingsalts and derivatives thereof, as well as various other medications usedto reduce the stabbing pains that may occur, including anticonvulsants(gabapentin, phenyloin, carbamazepine) or tricyclic antidepressants.Specific examples include acetaminophen, aspirin, amitriptyline(ELAVIL®), carbamazepine (TEGRETOL®), phenyltoin (DILANTIN®), gabapentin(NEURONTIN®), (E)-N-Vanillyl-8-methyl-6-noneamid (CAPSAICIN®), or anerve blocker.

“Corticosteroid” refers to any one of several synthetic or naturallyoccurring substances with the general chemical structure of steroidsthat mimic or augment the effects of the naturally occurringcorticosteroids. Examples of synthetic corticosteroids includeprednisone, prednisolone (including methylprednisolone), dexamethasonetriamcinolone, and betamethasone.

A “cancer vaccine,” as used herein is a composition that stimulates animmune response in a subject against a cancer. Cancer vaccines typicallyconsist of a source of cancer-associated material or cells (antigen)that may be autologous (from self) or allogenic (from others) to thesubject, along with other components (e.g., adjuvants) to furtherstimulate and boost the immune response against the antigen. Cancervaccines can result in stimulating the immune system of the subject toproduce antibodies to one or several specific antigens, and/or toproduce killer T cells to attack cancer cells that have those antigens.

“Cytotoxic radiotherapy” as used herein refers to radiation therapy thatinhibits or prevents the function of cells and/or causes destruction ofcells. Radiation therapy may include, for example, external beamirradiation or therapy with a radioactive labeled agent, such as anantibody. The term is intended to include use of radioactive isotopes(e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Bm¹⁵³, Bi²¹², Ra²²³, P³²,and radioactive isotopes of Lu).

A “subject” is a vertebrate, such as a mammal, e.g., a human. Mammalsinclude, but are not limited to, farm animals (such as cows), sportanimals, pets (such as cats, dogs and horses), primates, mice, and rats.

Except where indicated otherwise by context, the terms “first”polypeptide and “second” polypeptide, and variations thereof, are merelygeneric identifiers, and are not to be taken as identifying a specificor a particular polypeptide or component of antibodies of the invention.

Commercially available reagents referred to in the Examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following Examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., supra; Ausubel et al., Current Protocols inMolecular Biology (Green Publishing Associates and Wiley Interscience, NY, 1989); Innis et al., PCR Protocols: A Guide to Methods andApplications (Academic Press, Inc., NY, 1990); Harlow et al.,Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold SpringHarbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press, Oxford,1984); Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

II. Construction of Heteromultimeric Proteins

Typically, the heteromultimeric proteins described herein will comprisea significant portion of an antibody Fc region. In other aspects,however, the heavy chain comprises only a portion of the C_(H)1, C_(H)2,and/or C_(H)3 domains.

Heteromultimerization Domains

The heteromultimeric proteins comprise a heteromultimerization domain.To generate a substantially homogeneous population of heterodimericmolecule, the heterodimerization domain must have a strong preferencefor forming heterodimers over homodimers. Although the heteromultimericproteins exemplified herein use the knobs into holes technology tofacilitate heteromultimerization those skilled in the art willappreciate other heteromultimerization domains useful in the instantinvention.

Knobs into Holes

The use of knobs into holes as a method of producing multispecificantibodies is well known in the art. See U.S. Pat. No. 5,731,168 granted24 Mar. 1998 assigned to Genentech, PCT Pub. No. WO2009089004 published16 Jul. 2009 and assigned to Amgen, and US Pat. Pub. No. 20090182127published 16 Jul. 2009 and assigned to Novo Nordisk A/S. See also Marvinand Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann(2005) Acta Pharacol. Sin., 26:1-9. A brief discussion is provided here.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of a first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of a second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g. by alteringnucleic acid encoding the interface). Normally, nucleic acid encodingthe interface of the first polypeptide is altered to encode theprotuberance. To achieve this, the nucleic acid encoding at least one“original” amino acid residue in the interface of the first polypeptideis replaced with nucleic acid encoding at least one “import” amino acidresidue which has a larger side chain volume than the original aminoacid residue. It will be appreciated that there can be more than oneoriginal and corresponding import residue. The upper limit for thenumber of original residues which are replaced is the total number ofresidues in the interface of the first polypeptide. The side chainvolumes of the various amino residues are shown in the following table.

TABLE 1 Properties of Amino Acid Residues Accessible One-Letter SurfaceAbbre- MASS^(a) VOLUME^(b) Area^(c) Amino Acid viation (daltons)(Angstrom³) (Angstrom²) Alanine (Ala) A 71.08 88.6 115 Arginine (Arg) R156.20 173.4 225 Asparagine (Asn) N 114.11 117.7 160 Aspartic acid (Asp)D 115.09 111.1 150 Cysteine (Cys) C 103.14 108.5 135 Glutamine (Gln) Q128.14 143.9 180 Glutamic acid (Glu) E 129.12 138.4 190 Glycine (Gly) G57.06 60.1 75 Histidine (His) H 137.15 153.2 195 Isoleucine (Ile) I113.17 166.7 175 Leucine (Leu) L 113.17 166.7 170 Lysine (Lys) K 128.18168.6 200 Methionine (Met) M 131.21 162.9 185 Phenylalinine (Phe) F147.18 189.9 210 Proline (Pro) P 97.12 122.7 145 Serine (Ser) S 87.0889.0 115 Threonine (Thr) T 101.11 116.1 140 Tryptophan (Trp) W 186.21227.8 255 Tyrosine (Tyr) Y 163.18 193.6 230 Valine (Val) V 99.14 140.0155 ^(a)Molecular weight amino acid minus that of water. Values fromHandbook of Chemistry and Physics, 43rd ed. Cleveland, Chemical RubberPublishing Co., 1961. ^(b)Values from A. A. Zamyatnin, Prog. Biophys.Mol. Biol. 24: 107-123, 1972. ^(c)Values from C. Chothia, J. Mol. Biol.105: 1-14, 1975. The accessible surface area is defined in FIGS. 6-20 ofthis reference.

The preferred import residues for the formation of a protuberance aregenerally naturally occurring amino acid residues and are preferablyselected from arginine (R), phenylalanine (F), tyrosine (Y) andtryptophan (W). Most preferred are tryptophan and tyrosine. In oneembodiment, the original residue for the formation of the protuberancehas a small side chain volume, such as alanine, asparagine, asparticacid, glycine, serine, threonine or valine.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of a second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface of afirst polypeptide. The cavity may exist in the original interface or maybe introduced synthetically (e.g. by altering nucleic acid encoding theinterface). Normally, nucleic acid encoding the interface of the secondpolypeptide is altered to encode the cavity. To achieve this, thenucleic acid encoding at least one “original” amino acid residue in theinterface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue.The upper limit for the number of original residues which are replacedis the total number of residues in the interface of the secondpolypeptide. The side chain volumes of the various amino residues areshown in Table 1 above. The preferred import residues for the formationof a cavity are usually naturally occurring amino acid residues and arepreferably selected from alanine (A), serine (S), threonine (T) andvaline (V). Most preferred are serine, alanine or threonine. In oneembodiment, the original residue for the formation of the cavity has alarge side chain volume, such as tyrosine, arginine, phenylalanine ortryptophan.

An “original” amino acid residue is one which is replaced by an “import”residue which can have a smaller or larger side chain volume than theoriginal residue. The import amino acid residue can be a naturallyoccurring or non-naturally occurring amino acid residue, but preferablyis the former. “Naturally occurring” amino acid residues are thoseresidues encoded by the genetic code and listed in Table 1 above. By“non-naturally occurring” amino acid residue is meant a residue which isnot encoded by the genetic code, but which is able to covalently bindadjacent amino acid residue(s) in the polypeptide chain. Examples ofnon-naturally occurring amino acid residues are norleucine, ornithine,norvaline, homoserine and other amino acid residue analogues such asthose described in Ellman et al., Meth. Enzym. 202:301-336 (1991), forexample. To generate such non-naturally occurring amino acid residues,the procedures of Noren et al. Science 244: 182 (1989) and Ellman etal., supra can be used. Briefly, this involves chemically activating asuppressor tRNA with a non-naturally occurring amino acid residuefollowed by in vitro transcription and translation of the RNA. Themethod of the instant invention involves replacing at least one originalamino acid residue, but more than one original residue can be replaced.Normally, no more than the total residues in the interface of the firstor second polypeptide will comprise original amino acid residues whichare replaced. Typically, original residues for replacement are “buried”.By “buried” is meant that the residue is essentially inaccessible tosolvent. Generally, the import residue is not cysteine to preventpossible oxidation or mispairing of disulfide bonds.

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of afirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity relies on modeling the protuberance/cavity pair based upon athree-dimensional structure such as that obtained by X-raycrystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art.

By “original or template nucleic acid” is meant the nucleic acidencoding a polypeptide of interest which can be “altered” (i.e.genetically engineered or mutated) to encode a protuberance or cavity.The original or starting nucleic acid may be a naturally occurringnucleic acid or may comprise a nucleic acid which has been subjected toprior alteration (e.g. a humanized antibody fragment). By “altering” thenucleic acid is meant that the original nucleic acid is mutated byinserting, deleting or replacing at least one codon encoding an aminoacid residue of interest. Normally, a codon encoding an original residueis replaced by a codon encoding an import residue. Techniques forgenetically modifying a DNA in this manner have been reviewed inMutagenesis: a Practical Approach, M. J. McPherson, Ed., (IRL Press,Oxford, UK. (1991), and include site-directed mutagenesis, cassettemutagenesis and polymerase chain reaction (PCR) mutagenesis, forexample. By mutating an original/template nucleic acid, anoriginal/template polypeptide encoded by the original/template nucleicacid is thus correspondingly altered.

The protuberance or cavity can be “introduced” into the interface of afirst or second polypeptide by synthetic means, e.g. by recombinanttechniques, in vitro peptide synthesis, those techniques for introducingnon-naturally occurring amino acid residues previously described, byenzymatic or chemical coupling of peptides or some combination of thesetechniques. Accordingly, the protuberance or cavity which is“introduced” is “non-naturally occurring” or “non-native”, which meansthat it does not exist in nature or in the original polypeptide (e.g. ahumanized monoclonal antibody).

Generally, the import amino acid residue for forming the protuberancehas a relatively small number of “rotamers” (e.g. about 3-6). A“rotomer” is an energetically favorable conformation of an amino acidside chain. The number of rotomers of the various amino acid residuesare reviewed in Ponders and Richards, J. Mol. Biol. 193: 775-791 (1987).

III. Vectors, Host Cells and Recombinant Methods

For recombinant production of a heteromultimeric protein (e.g., anantibody) of the invention, the nucleic acid encoding it is isolated andinserted into a replicable vector for further cloning (amplification ofthe DNA) or for expression. DNA encoding the antibody is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The choice of vector depends in part on the host cell to beused. Generally, preferred host cells are of either prokaryotic oreukaryotic (generally mammalian, but also including fungi (e.g., yeast),insect, plant, and nucleated cells from other multicellular organisms)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

a. Generating Heteromultimeric Proteins Using Prokaryotic Host Cells

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of theheteromultimeric proteins (e.g., an antibody) of the invention can beobtained using standard recombinant techniques. Desired polynucleotidesequences may be isolated and sequenced from, for example, antibodyproducing cells such as hybridoma cells. Alternatively, polynucleotidescan be synthesized using nucleotide synthesizer or PCR techniques. Onceobtained, sequences encoding the polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in prokaryotic hosts. Many vectors that are availableand known in the art can be used for the purpose of the presentinvention. Selection of an appropriate vector will depend mainly on thesize of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM.TM.-11 may be utilized in making arecombinant vector which can be used to transform susceptible host cellssuch as E. coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. An inducible promoteris a promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g., the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding, for example, the light or heavy chain by removingthe promoter from the source DNA via restriction enzyme digestion andinserting the isolated promoter sequence into the vector of theinvention. Both the native promoter sequence and many heterologouspromoters may be used to direct amplification and/or expression of thetarget genes. In some embodiments, heterologous promoters are utilized,as they generally permit greater transcription and higher yields of theexpressed target gene as compared to the native target polypeptidepromoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker to operably ligate them to cistrons encoding the genes of theheteromultimeric protein, e.g., the target light and heavy chains(Siebenlist et al., (1980) Cell 20: 269), using linkers or adaptors tosupply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the signal sequences native to theheterologous polypeptides, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the groupconsisting of the alkaline phosphatase, penicillinase, Ipp, orheat-stable enterotoxin II (STII) leaders, LamB, PhoE, PeIB, OmpA andMBP. In one embodiment of the invention, the signal sequences used inboth cistrons of the expression system are STII signal sequences orvariants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxastrains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. See Proba and Pluckthun Gene, 159:203(1995).

Prokaryotic host cells suitable for expressing heteromultimeric proteins(e.g., antibodies) of the invention include Archaebacteria andEubacteria, such as Gram-negative or Gram-positive organisms. Examplesof useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g.,B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa),Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus,Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment,gram-negative cells are used. In one embodiment, E. coli cells are usedas hosts for the invention. Examples of E. coli strains include strainW3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington,D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCCDeposit No. 27,325) and derivatives thereof, including strain 33D3having genotype W3110 AfhuA (AtonA) ptr3 lac iq iacL8 ΔompTΔ(nmpc-fepE)degP41 kan^(R) (U.S. Pat. No. 5,639,635). Other strains and derivativesthereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli _(λ)1776(ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. In oneembodiment, E. coli App finds particular use. These examples areillustrative rather than limiting. Methods for constructing derivativesof any of the above-mentioned bacteria having defined genotypes areknown in the art and described in, for example, Bass et al., Proteins,8:309-314 (1990). It is generally necessary to select the appropriatebacteria taking into consideration replicability of the replicon in thecells of a bacterium. For example, E. coli, Serratia, or Salmonellaspecies can be suitably used as the host when well known plasmids suchas pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.Typically the host cell should secrete minimal amounts of proteolyticenzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

ii. Polypeptide Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include Luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the first and second hinge-containing host cells arecultured separately and the expressed polypeptides of the presentinvention are secreted into and recovered from the periplasm of the hostcells separately. In a second embodiment, the first and secondhinge-containing host cells are cultured separately and prior to theisolation of the hinge-containing polypeptides, the two host cellcultures are mixed together and the cells pelleted. In a thirdembodiment, the first and second hinge-containing host cells arecultured separately, centrifuged and resuspended separately and thenmixed together prior to isolation of the hinge-containing polypeptides.In fourth embodiment, the first and second hinge-containing host cellsare cultured together in the same culture vessel. Protein recoverytypically involves disrupting the microorganism cell membrane, generallyby such means as osmotic shock, sonication or lysis. Once cells aredisrupted, cell debris or whole cells may be removed by centrifugationor filtration. The proteins may be further purified, for example, byaffinity resin chromatography. Alternatively, proteins can betransported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay. The isolated polypeptides will be used toproduce the heteromultimeric proteins at

In one aspect of the invention, heteromultimeric protein (e.g.,antibody) production is conducted in large quantity by a fermentationprocess. Various large-scale fed-batch fermentation procedures areavailable for production of recombinant proteins. Large-scalefermentations have at least 1000 liters of capacity, preferably about1,000 to 100,000 liters of capacity. These fermentors use agitatorimpellers to distribute oxygen and nutrients, especially glucose (thepreferred carbon/energy source). Small scale fermentation refersgenerally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secretedheteromultimeric proteins (e.g., antibodies), additional vectorsoverexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB,DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isomerasewith chaperone activity) can be used to co-transform the hostprokaryotic cells. The chaperone proteins have been demonstrated tofacilitate the proper folding and solubility of heterologous proteinsproduced in bacterial host cells. Chen et al. (1999) J Bio Chem274:19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou etal., U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol.Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),Proc. Natl. Acad. Sci. USA 95:2773-2777; Georgiou et al., U.S. Pat. No.5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al.,Microbial Drug Resistance, 2:63-72 (1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention. In asecond embodiment, the E. coli strain is deficient for a lipoprotein ofthe outer membrane (Δlpp).

iii. Heteromultimeric Protein Purification

In one embodiment, the heteromultimeric protein produced herein isfurther purified to obtain preparations that are substantiallyhomogeneous for further assays and uses. Standard protein purificationmethods known in the art can be employed. The following procedures areexemplary of suitable purification procedures: fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase H PLC, chromatography on silica or on a cation-exchange resin suchas DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, andgel filtration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of, for example, full length antibodyproducts of the invention. Protein A is a 41 kD cell wall protein fromStaphylococcus aureus which binds with a high affinity to the Fc regionof antibodies. Lindmark et al. (1983) J. Immunol. Meth. 62:1-13. Thesolid phase to which Protein A is immobilized is preferably a columncomprising a glass or silica surface, more preferably a controlled poreglass column or a silicic acid column. In some applications, the columnhas been coated with a reagent, such as glycerol, in an attempt toprevent nonspecific adherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. The heteromultimeric protein(e.g., antibody) is recovered from the solid phase by elution.

b. Generating Heteromultimeric Proteins Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

i. Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available. The DNA for such precursor region is ligated inreading frame to DNA encoding the desired heteromultimeric protein(s)(e.g., antibodies).

ii. Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used, but only because it contains the early promoter.

iii. Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand —II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See, for example, U.S. Pat. No. 4,965,199.

iv. Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the desiredhinge-containing polypeptide(s) (e.g., antibody) nucleic acid. Promotersequences are known for eukaryotes. Virtually all eukaryotic genes havean AT-rich region located approximately 25 to 30 bases upstream from thesite where transcription is initiated. Another sequence found 70 to 80bases upstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Desired hinge-containing polypeptide(s) (e.g., antibody) transcriptionfrom vectors in mammalian host cells is controlled, for example, bypromoters obtained from the genomes of viruses such as, for example,polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, or from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

v. Enhancer Element Component

Transcription of DNA encoding the desired hinge-containingpolypeptide(s) (e.g., antibody) by higher eukaryotes can be increased byinserting an enhancer sequence into the vector. Many enhancer sequencesare now known from mammalian genes (e.g., globin, elastase, albumin,α-fetoprotein, and insulin genes). Also, one may use an enhancer from aeukaryotic cell virus. Examples include the SV40 enhancer on the lateside of the replication origin (bp 100-270), the cytomegalovirus earlypromoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. See also Yaniv, Nature297:17-18 (1982) for a description of elements for enhancing activationof eukaryotic promoters. The enhancer may be spliced into the vector ata position 5′ or 3′ to the antibody polypeptide-encoding sequence,provided that enhancement is achieved, but is generally located at asite 5′ from the promoter.

vi. Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

vii. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for desired hinge-containing polypeptide(s) (e.g.,antibody) production and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences.

viii. Culturing the host cells

The host cells used to produce a desired hinge-containing polypeptide(s)(e.g., antibody) of this invention may be cultured in a variety ofmedia. Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used asculture media for the host cells. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

ix. Purification of Heteromultimeric Proteins

When using recombinant techniques, the hinge-containing polypeptides canbe produced intracellularly, or directly secreted into the medium. Ifthe hinge-containing polypeptide is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Where thehinge-containing polypeptide is secreted into the medium, supernatantsfrom such expression systems are generally first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. A protease inhibitorsuch as PMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The heteromultimer composition prepared from the cells can be purifiedusing, for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt). The production of the heteromultimericproteins can alternatively or additionally (to any of the foregoingparticular methods) comprise dialyzing a solution comprising a mixtureof the polypeptides.

x. Antibody Production Using Baculovirus

Recombinant baculovirus may be generated by co-transfecting a plasmidencoding an antibody or antibody fragment and BaculoGold™ virus DNA(Pharmingen) into an insect cell such as a Spodoptera frugiperda cell(e.g., Sf9 cells; ATCC CRL 1711) or a Drosophila melanogaster S2 cellusing, for example, lipofectin (commercially available from GIBCO-BRL).In a particular example, an antibody sequence is fused upstream of anepitope tag contained within a baculovirus expression vector. Suchepitope tags include poly-His tags. A variety of plasmids may beemployed, including plasmids derived from commercially availableplasmids such as pVL1393 (Novagen) or pAcGP67B (Pharmingen). Briefly,the sequence encoding an antibody or a fragment thereof may be amplifiedby PCR with primers complementary to the 5′ and 3′ regions. The 5′primer may incorporate flanking (selected) restriction enzyme sites. Theproduct may then be digested with the selected restriction enzymes andsubcloned into the expression vector.

After transfection with the expression vector, the host cells (e.g., Sf9cells) are incubated for 4-5 days at 28° C. and the released virus isharvested and used for further amplifications. Viral infection andprotein expression may be performed as described, for example, byO'Reilley et al. (Baculovirus expression vectors: A Laboratory Manual.Oxford: Oxford University Press (1994)).

Expressed poly-His tagged antibody can then be purified, for example, byNi2+-chelate affinity chromatography as follows. Extracts can beprepared from recombinant virus-infected Sf9 cells as described byRupert et al. (Nature 362:175-179 (1993)). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL HEPES pH 7.9; 12.5 mMMgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate; 300 mM NaCl; 10% glycerol pH 7.8) and filtered througha 0.45 μm filter. A Ni2+-NTA agarose column (commercially available fromQiagen) is prepared with a bed volume of 5 mL, washed with 25 mL ofwater, and equilibrated with 25 mL of loading buffer. The filtered cellextract is loaded onto the column at 0.5 mL per minute. The column iswashed to baseline A280 with loading buffer, at which point fractioncollection is started. Next, the column is washed with a secondary washbuffer (50 mM phosphate; 300 mM NaCl; 10% glycerol pH 6.0), which elutesnonspecifically bound protein. After reaching A280 baseline again, thecolumn is developed with a 0 to 500 mM Imidazole gradient in thesecondary wash buffer. One mL fractions are collected and analyzed bySDS-PAGE and silver staining or Western blot with Ni2+-NTA-conjugated toalkaline phosphatase (Qiagen). Fractions containing the elutedHis10-tagged antibody are pooled and dialyzed against loading buffer.

Alternatively, purification of the antibody can be performed using knownchromatography techniques, including for instance, Protein A or proteinG column chromatography. In one embodiment, the antibody of interest maybe recovered from the solid phase of the column by elution into asolution containing a chaotropic agent or mild detergent. Exemplarychaotropic agents and mild detergents include, but are not limited to,Guanidine-HCl, urea, lithium perclorate, Arginine, Histidine, SDS(sodium dodecyl sulfate), Tween, Triton, and NP-40, all of which arecommercially available.

IV. Heteromultimeric Protein Formation/Assembly

The formation of the complete heteromultimeric protein involves thereassembly of the first and second hinge-containing polypeptides bydisulfide bond formation which in the present invention is referred toas refolding. Refolding includes the association of the firsthinge-containing polypeptide with the second hinge-containingpolypeptide and the formation of the interchain disulfide bonds.Refolding, also termed renaturing, in the present invention is done invitro without the addition of reductant.

The host cells may be cultured using the above described methods eitheras separate cultures or as a single culture. In one method, the firsthost cells and second host cells are grown in the same culture vessel(sometimes referred to herein as co-cultured or a mixed culture). Inanother method, the first and second host cells are grown in separateculture vessels. In one method, the separate cultures are processedseparately then mixed/combined prior to disruption of the cellularmembrane. In another method, the separate cultures are mixed thenprocessed prior to disruption of the cellular membrane. In one method,the separate cultures are mixed without further processing prior todisruption of the cellular membrane. In one method, the single culturecomprising the first and second host cells is processed prior todisruption of the cellular membrane. In another method, the co-culturedcells are not processed prior to disruption of the cellular membrane.Processing of the cells comprises centrifugation and resuspension in anappropriate buffer (e.g., extraction buffer).

Extraction buffers are known in the art and the skilled artisan will beable to determine which buffer to use without undue experimentation.

The host cell membranes are disrupted using methods known in the art.Such methods include cell membrane permeablization and cell membranedisintegration. Permeablizing the cell membrane refers to rendering themembrane “leaky”, e.g., by introducing holes, without destroying theoverall integrity of the membrane such that the cell remains viable. Inother words, permeabilization provides macromolecular movement acrossthe cellular membrane and preserves cellular structure sufficiently toallow continued cell viability. In contrast, cell membranedisintegration results in the cellular contents being released into theextracellular milieu and cell death.

Methods for disrupting cell membranes include but are not limited toenzymatic lysis. sonication, osmotic shock, passage through amicrofluidizer, addition of EDTA, use various detergents, solvents (suchas toluene, dimethyl sulfoxide, etc), surfactants (such as Triton-X 100,Tween 20, etc), hypotonic buffers, use of freeze/thaw techniques,electroporation, and passage through a stainless steel ball homogenizer.

Once the hinge-containing polypeptides are released from the cell(either by permeabilization or disintegration) the heteromultimerizationdomains will drive the association of the heteromultimeric proteins.Inter-chain disulfide formation of the associated hinge-containingpolypeptides proceeds without the addition of reducing agents. Theresultant disulfide linked heteromultimeric protein is then purified.Optionally, it may be formulated for research, diagnostic, therapeuticor other purposes.

V. Target Molecules

Examples of molecules that may be targeted by a heteromultimeric proteinof this invention include, but are not limited to, soluble serumproteins and their receptors and other membrane bound proteins (e.g.,adhesins).

In another embodiment the heteromultimeric protein of the invention iscapable of binding one, two or more cytokines, cytokine-relatedproteins, and cytokine receptors selected from the group consisting ofBMPI, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, CSFI (M-CSF), CSF2(GM-CSF), CSF3 (G-CSF), EPO, FGFI (aFGF), FGF2 (bFGF), FGF3 (int-2),FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12,FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2,IFNAI, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNBI, IFNG, IFNWI, FELI, FELI(EPSELON), FELI (ZETA), ILA, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8,IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B,IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B,IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB,TNF (TNF-a), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL),TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand),TNFSFIO (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April),TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF,VEGFB, VEGFC, ILIR1, IL1R2, IL1RL1, LL1RL2, IL2RA, IL2RB, IL2RG, IL3RA,IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, ILIORA, ILIORB, IL1RA,IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA,IL21R, IL22R, IL1HY1, ILRAP, IL1RAPL1, IL1RAPL2, ILRN, IL6ST, IL18BP,IL18RAP, IL22RA2, AIFI, HGF, LEP (leptin), PTN, and THPO.

In another embodiment, a target molecule is a chemokine, chemokinereceptor, or a chemokine-related protein selected from the groupconsisting of CCLI (I-309), CCL2 (MCP-1/MCAF), CCL3 (MIP-Ia), CCL4(MIP-Ib), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCLH (eotaxin),CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18(PARC), CCL19 (MDP-3b), CCL20 (MIP-3a), CCL21 (SLC/exodus-2), CCL22(MDC/STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK),CCL26 (eotaxin-3), CCL27 (CTACK/ILC), CCL28, CXCLI (GROI), CXCL2 (GRO2),CXCL3 (GRO3), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP10), CXCL11 (I-TAC), CXCL12 (SDFI), CXCL13, CXCL14, CXCL16, PF4 (CXCL4),PPBP (CXCL7), CX3CL1 (SCYDI), SCYEI, XCLI (lymphotactin), XCL2 (SCM-Ib),BLRI (MDR15), CCBP2 (D6/JAB61), CCRI (CKRI/HM145), CCR2 (mcp-IRB/RA),CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6(CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBII), CCR8(CMKBR8/TERI/CKR-LI), CCR9 (GPR-9-6), CCRLI (VSHKI), CCRL2 (L-CCR), XCRI(GPR5/CCXCRI), CMKLRI, CMKORI (RDCI), CX3CR1 (V28), CXCR4, GPR2 (CCRIO),GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6 (TYMSTR/STRL33/Bonzo),HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R (GPR16), TCPIO, CKLFSF2,CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3,GRCCIO (CIO), EPO, FY (DARC), GDF5, HDFIA, DL8, PRL, RGS3, RGS13, SDF2,SLIT2, TLR2, TLR4, TREMI, TREM2, and VHL.

In another embodiment the heteromultimeric proteins of the invention arecapable of binding one or more targets selected from the groupconsisting of ABCFI; ACVRI; ACVRIB; ACVR2; ACVR2B; ACVRLI; ADORA2A;Aggrecan; AGR2; AICDA; AIFI; AIGI; AKAPI; AKAP2; AMH; AMHR2; ANGPTI;ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOCI; AR; AZGPI(zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF (BLys); BAGI; BAII; BCL2;BCL6; BDNF; BLNK; BLRI (MDR15); BMPI; BMP2; BMP3B (GDFIO); BMP4; BMP6;BMP8; BMPRIA; BMPRIB; BMPR2; BPAGI (plectin); BRCAI; C19orflO (IL27w);C3; C4A; C5; C5R1; CANTI; CASP1; CASP4; CAVI; CCBP2 (D6/JAB61); CCLI(1-309); CCLII (eotaxin); CCL13 (MCP-4); CCL15 (MIP-Id); CCL16 (HCC-4);CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20(MIP-3a); CCL21 (MTP-2); SLC; exodus-2; CCL22 (MDC/STC-I); CCL23(MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3);CCL27 (CTACK/ILC); CCL28; CCL3 (MTP-Ia); CCL4 (MDP-Ib); CCL5 (RANTES);CCL7 (MCP-3); CCL8 (mcp-2); CCNAI; CCNA2; CCNDI; CCNEI; CCNE2; CCRI(CKRI/HM145); CCR2 (mcp-IRB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5(CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBII);CCR8 (CMKBR8/TERI/CKR-LI); CCR9 (GPR-9-6); CCRLI (VSHKI); CCRL2 (L-CCR);CD164; CD19; CDIC; CD20; CD200; CD22; CD24; CD28; CD3; CD37; CD38; CD3E;CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74;CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDHI (E-cadherin); CDH10;CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3;CDK4; CDK5; CDK6; CDK7; CDK9; CDKNIA (p21WapI/CipI); CDKNIB (p27KipI);CDKNIC; CDKN2A (P16NK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CERI; CHGA;CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6;CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin);CMKLRI; CMKORI (RDCI); CNRI; COL18A1; COLIAI; COL4A3; COL6A1; CR2; CRP;CSFI (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNBI (b-catenin);CTSB (cathepsin B); CX3CL1 (SCYDI); CX3CR1 (V28); CXCLI (GROI); CXCL10(IP-10); CXCLII (I-TAC/IP-9); CXCL12 (SDFI); CXCL13; CXCL14; CXCL16;CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9(MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5;CYCI; CYSLTRI; DAB2IP; DES; DKFZp451J0118; DNCLI; DPP4; E2F1; ECGFI;EDGI; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3;EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESRI; ESR2; F3 (TF); FADD; FasL;FASN; FCERIA; FCER2; FCGR3A; FGF; FGFI (aFGF); FGF10; FGF11; FGF12;FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20;FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7(KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FELI (EPSILON); FILI (ZETA);FLJ12584; FLJ25530; FLRTI (fibronectin); FLTI; FOS; FOSLI (FRA-I); FY(DARC); GABRP (GABAa); GAGEBI; GAGECI; GALNAC4S-6ST; GATA3; GDF5; GFI1;GGT1; GM-CSF; GNASI; GNRHI; GPR2 (CCRIO); GPR31; GPR44; GPR81 (FKSG80);GRCCIO (CIO); GRP; GSN (Gelsolin); GSTPI; HAVCR2; HDAC4; HDAC5; HDAC7A;HDAC9; HGF; HIFIA; HDPI; histamine and histamine receptors; HLA-A;HLA-DRA; HM74; HMOXI; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNAI;IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; DFNWI; IGBPI; IGFI;IGFIR; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11;IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2;IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP;IL18R1; IL18RAP; IL19; IL1A; IL1B; ILIF10; IL1F5; IL1F6; IL1F7; IL1F8;IL1F9; IL1HYI; IL1RI; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2,ILIRN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25;IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA;IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); EL7; EL7R;EL8; IL8RA; DL8RB; IL8RB; DL9; DL9R; DLK; INHA; INHBA; INSL3; INSL4;IRAKI; ERAK2; ITGAI; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3;ITGB4 (b 4 integrin); JAGI; JAKI; JAK3; JUN; K6HF; KAII; KDR; KITLG;KLF5 (GC Box BP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4;KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KHTHB6 (hair-specifictype H keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA(TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp;MAP2K7 (c-Jun); MDK; MIBI; midkine; MEF; MIP-2; MK167; (Ki-67); MMP2;MMP9; MS4A1; MSMB; MT3 (metallothionectin-Ill); MTSSI; MUCI (mucin);MYC; MYD88; NCK2; neurocan; NFKBI; NFKB2; NGFB (NGF); NGFR; NgR-Lingo;NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NMEI (NM23A); NOX5; NPPB; NROBI;NROB2; NRIDI; NR1D2; NR1H2; NR1H3; NR1H4; NR112; NR113; NR2C1; NR2C2;NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3;NR5A1; NR5A2; NR6A1; NRPI; NRP2; NT5E; NTN4; ODZI; OPRDI; P2RX7; PAP;PARTI; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAMI; PF4 (CXCL4); PGF;PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDCI; PPBP (CXCL7);PPID; PRI; PRKCQ; PRKDI; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN;PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGSI; RGS13; RGS3; RNFIIO(ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin2);SCGB2A2 (mammaglobin 1); SCYEI (endothelial Monocyte-activatingcytokine); SDF2; SERPINAI; SERPINA3; SERP1NB5 (maspin); SERPINEI(PAl-1); SERPDMF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPPI;SPRRIB (Sprl); ST6GAL1; STABI; STAT6; STEAP; STEAP2; TB4R2; TBX21;TCPIO; TDGFI; TEK; TGFA; TGFBI; TGFBIII; TGFB2; TGFB3; TGFBI; TGFBRI;TGFBR2; TGFBR3; THIL; THBSI (thrombospondin-1); THBS2; THBS4; THPO; TIE(Tie-1); TMP3; tissue factor; TLRIO; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7;TLR8; TLR9; TNF; TNF-α; TNFAEP2 (B94); TNFAIP3; TNFRSFIIA; TNFRSFIA;TNFRSFIB; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9;TNFSFIO (TRAIL); TNFSFI 1 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April);TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand);TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-likereceptors; TOP2A (topoisomerase Ea); TP53; TPMI; TPM2; TRADD; TRAFI;TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREMI; TREM2; TRPC6; TSLP; TWEAK;VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCLI (lymphotactin); XCL2(SCM-Ib); XCRI(GPR5/CCXCRI); YYI; and ZFPM2.

Preferred molecular target molecules for antibodies encompassed by thepresent invention include CD proteins such as CD3, CD4, CD8, CD16, CD19,CD20, CD34; CD64, CD200 members of the ErbB receptor family such as theEGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules suchas LFA-1, Maci, p150.95, VLA-4, ICAM-1, VCAM, alpha4/beta7 integrin, andalphav/beta3 integrin including either alpha or beta subunits thereof(e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factorssuch as VEGF-A, VEGF-C; tissue factor (TF); alpha interferon(QalphalFN); TNFalpha, an interleukin, such as IL-1beta, IL-3, IL-4,IL-5, IL-8, IL-9, IL-13, IL17A/F, IL-18, IL-13Ralpha1, IL13Ralpha2,IL-4R, IL-5R, IL-9R, IgE; blood group antigens; flk2/flt3 receptor;obesity (OB) receptor; mpl receptor; CTLA-4; RANKL, RANK, RSV F protein,protein C etc.

In one embodiment, the heteromultimeric proteins of this invention bindlow-density lipoprotein receptor-related protein (LRP)-1 or LRP-8 ortransferrin receptor, and at least one target selected from the groupconsisting of 1) beta-secretase (BACE1 or BACE2), 2) alpha-secretase, 3)gamma-secretase, 4) tau-secretase, 5) amyloid precursor protein (APP),6) death receptor 6 (DR6), 7) amyloid beta peptide, 8) alpha-synuclein,9) Parkin, 10) Huntingtin, 11) p75 NTR, and 12) caspase-6.

In one embodiment, the heteromultimeric proteins of this invention bindsto at least two target molecules selected from the group consisting of:IL-1alpha and IL-1beta, IL-12 and IL-18; IL-13 and IL-9; IL-13 and IL-4;IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-1beta; IL-13 and IL-25;IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-p; IL-13 andLHR agonist; IL-12 and TWEAK, IL-13 and CL25; IL-13 and SPRR2a; IL-13and SPRR2b; IL-13 and ADAM8, IL-13 and PED2, IL17A and IL17F, CD3 andCD19, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD38and CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20; CD-8 and IL-6;CD20 and BR3, TNFalpha and TGF-beta, TNFalpha and IL-1beta; TNFalpha andIL-2, TNF alpha and IL-3, TNFalpha and IL-4, TNFalpha and IL-5, TNFalphaand IL6, TNFalpha and IL8, TNFalpha and IL-9, TNFalpha and IL-10,TNFalpha and IL-11, TNFalpha and IL-12, TNFalpha and IL-13, TNFalpha andIL-14, TNFalpha and IL-15, TNFalpha and IL-16, TNFalpha and IL-17,TNFalpha and IL-18, TNFalpha and IL-19, TNFalpha and IL-20, TNFalpha andIL-23, TNFalpha and IFNalpha, TNFalpha and CD4, TNFalpha and VEGF,TNFalpha and MIF, TNFalpha and ICAM-1, TNFalpha and PGE4, TNFalpha andPEG2, TNFalpha and RANK ligand. TNFalpha and Te38; TNFalpha and BAFF;TNFalpha and CD22; TNFalpha and CTLA-4; TNFalpha and GP130; TNFα andIL-12p40; VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 andHER2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8,VEGF and MET, VEGFR and MET receptor, VEGFR and EGFR, HER2 and CD64,HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR(HER1) and HER2, EGFRand HER3, EGFR and HER4, IL-13 and CD40L, IL4 and CD40L, TNFR1 andIL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG andCD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTNO2; IGF1 and IGF2;IGF1/2 and Erb2B; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGpand RGM A; PDL- and CTLA-4; and RGM A and RGM B.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g.,the extracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen.

Such cells can be derived from a natural source (e.g., cancer celllines) or may be cells which have been transformed by recombinanttechniques to express the transmembrane molecule. Other antigens andforms thereof useful for preparing antibodies will be apparent to thosein the art.

VI. Activity Assays

The heteromultimeric proteins of the present invention can becharacterized for their physical/chemical properties and biologicalfunctions by various assays known in the art.

The purified heteromultimeric proteins can be further characterized by aseries of assays including, but not limited to, N-terminal sequencing,amino acid analysis, non-denaturing size exclusion high pressure liquidchromatography (H PLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the immunoglobulins producedherein are analyzed for their biological activity. In some embodiments,the immunoglobulins of the present invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include, without limitation, any director competitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immnosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. An illustrative antigen binding assay isprovided below in the Examples section.

In one embodiment, the present invention contemplates an alteredantibody that possesses some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the produced heteromultimeric proteinare measured to ensure that only the desired properties are maintained.In vitro and/or in vivo cytotoxicity assays can be conducted to confirmthe reduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theheteromultimeric protein lacks FcγR binding (hence likely lacking ADCCactivity), but retains FcRn binding ability. The primary cells formediating ADCC, NK cells, express Fc(RIII only, whereas monocytesexpress Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). An example of an in vitro assay to assess ADCCactivity of a molecule of interest is described in U.S. Pat. No.5,500,362 or 5,821,337. Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. To assess complement activation,a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol.Methods 202:163 (1996), may be performed. FcRn binding and in vivoclearance/half life determinations can also be performed using methodsknown in the art.

VII. Conjugated Proteins

The invention also provides conjugated proteins such as conjugatedantibodies or immunoconjugates (for example, “antibody-drug conjugates”or “ADC”), comprising any of the heteromultimeric proteins describedherein (e.g., an antibody made according to the methods describedherein) where one of the constant regions of the light chain or theheavy chain is conjugated to a chemical molecule such as a dye orcytotoxic agent such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate). In particular, asdescribed herein, the use of heteromultimerization domains enables theconstruction of antibodies containing two different heavy chains (HC1and HC2) as well as two different light chains (LC1 and LC2). Animmunoconjugate constructed using the methods described herein maycontain the cytotoxic agent conjugated to a constant region of only oneof the heavy chains (HC1 or HC2) or only one of the light chains (LC1 orLC2). Also, because the immunoconjugate can have the cytotoxic agentattached to only one heavy or light chain, the amount of the cytotoxicagent being administered to a subject is reduced relative toadministration of an antibody having the cytotoxic agent attached toboth heavy or light chains. Reducing the amount of cytotoxic agent beingadministered to a subject limits adverse side effects associated withthe cytotoxic agent.

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e., drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos, Anticancer Research19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev.26:151-172 (1997); U.S. Pat. No. 4,975,278) allows targeted delivery ofthe drug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., Lancet (Mar. 15,1986):603-605 (1986); Thorpe, (1985) “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84:Biological And Clinical Applications, A. Pinchera et al. (eds.), pp.475-506). Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., Cancer Immunol. Immunother.21:183-187 (1986)). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al., Jour. of the Nat. Cancer Inst.92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters10:1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791(2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci.USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (e.g., above). Enzymatically active toxins andfragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ¹²²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

i. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) of the invention conjugated to one or moremaytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. Patent ApplicationPublication No. 2005/0169933, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. Patent Application Publication No. 2005/0169933. The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred. Additional linking groups are described andexemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

ii. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483 and 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al., Antimicrob. Agents and Chemother. 45(12):3580-3584(2001)) and have anticancer (U.S. Pat. No. 5,663,149) and antifungalactivity (Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965(1998)). The dolastatin or auristatin drug moiety may be attached to theantibody through the N- (amino) terminus or the C- (carboxyl) terminusof the peptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Application Publication No. 2005/0238649, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lubke, “The Peptides,”volume 1, pp. 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. Nos. 5,635,483 and5,780,588; Pettit et al., J. Nat. Prod. 44:482-485 (1981); Pettit etal., Anti-Cancer Drug Design 13:47-66 (1998); Poncet, Curr. Pharm. Des.5:139-162 (1999); and Pettit, Fortschr. Chem. Org. Naturst. 70:1-79(1997). See also Doronina, Nat. Biotechnol. 21(7):778-784 (2003); and“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Application Publication No. 2005/0238649, hereby incorporated byreference in its entirety (disclosing, e.g., linkers and methods ofpreparing monomethylvaline compounds such as MMAE and MMAF conjugated tolinkers).

iii. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, and 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-y₁ ^(I), PSAG and θ^(I)₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug that the antibody can beconjugated is QFA, which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

iv. Other cytotoxic agents

Other antitumor agents that can be conjugated to the antibodies of theinvention or made according to the methods described herein includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394 and 5,770,710, as well as esperamicins (U.S. Pat. No.5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes (see, for example, WO 93/21232, publishedOct. 28, 1993).

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of a tumor, the antibody may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², F³², Pb²¹² and radioactiveisotopes of Lu. When the conjugate is used for detection, it maycomprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys.Res. Commun. 80:49-57 (1978)) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.The linker may be a “cleavable linker” facilitating release of thecytotoxic drug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

v. Preparation of Conjugated Antibodies

In the conjugated antibodies of the invention, an antibody is conjugatedto one or more moieties (for example, drug moieties), e.g., about 1 toabout 20 moieties per antibody, optionally through a linker. Theconjugated antibodies may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including: (1) reaction of a nucleophilic group ofan antibody with a bivalent linker reagent via a covalent bond, followedby reaction with a moiety of interest; and (2) reaction of anucleophilic group of a moiety with a bivalent linker reagent via acovalent bond, followed by reaction with the nucleophilic group of anantibody. Additional methods for preparing conjugated antibodies aredescribed herein.

The linker reagent may be composed of one or more linker components.Exemplary linker components include 6-maleimidocaproyl (“MC”),maleimidopropanoyl (“MP”), valine-citrulline (“val-cit”),alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”),N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Application Publication No. 2005/0238649, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g., lysine,(iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxylor amino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.,cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Conjugated antibodies of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drugor other moiety. The sugars of glycosylated antibodies may be oxidized,e.g., with periodate oxidizing reagents, to form aldehyde or ketonegroups which may react with the amine group of linker reagents or drugor other moieties. The resulting imine Schiff base groups may form astable linkage, or may be reduced, e.g., by borohydride reagents to formstable amine linkages. In one embodiment, reaction of the carbohydrateportion of a glycosylated antibody with either glactose oxidase orsodium meta-periodate may yield carbonyl (aldehyde and ketone) groups inthe protein that can react with appropriate groups on the drug or othermoiety (Hermanson, Bioconjugate Techniques). In another embodiment,proteins containing N-terminal serine or threonine residues can reactwith sodium meta-periodate, resulting in production of an aldehyde inplace of the first amino acid (Geoghegan and Stroh, Bioconjugate Chem.3:138-146 (1992); U.S. Pat. No. 5,362,852). Such aldehyde can be reactedwith a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a moiety (such as a drug moiety)include, but are not limited to: amine, thiol, hydroxyl, hydrazide,oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide groups capable of reacting to form covalent bonds withelectrophilic groups on linker moieties and linker reagents including:(i) active esters such as NHS esters, HOBt esters, haloformates, andacid halides; (ii) alkyl and benzyl halides such as haloacetamides; and(iii) aldehydes, ketones, carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate. In yet another embodiment, the antibody maybe conjugated to a “receptor” (such streptavidin) for utilization intumor pre-targeting wherein the antibody-receptor conjugate isadministered to the individual, followed by removal of unbound conjugatefrom the circulation using a clearing agent and then administration of a“ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g.,a radionucleotide).

VIII. Utility

The present methods provided for herein find industrial applicability inthe production of heteromultimeric proteins. The inventive methodsreduce the amount of work involved in two separate fermentation andisolations as are technical difficulties inherent in two separatefermentations. Furthermore, elimination of the annealment and redoxsteps of the prior methods procedures can increase yields and decreaseprocessing complexity and costs.

The heteromultimeric proteins described herein find use in, for example,in vitro, ex vivo and in vivo therapeutic methods. The inventionprovides various methods based on using one or more of these molecules.In certain pathological conditions, it is necessary and/or desirable toutilize heteromultimeric proteins, e.g., multispecific antibodies. Theinvention provides these heteromultimeric proteins, which can be usedfor a variety of purposes, for example as therapeutics, prophylacticsand diagnostics. For example, the invention provides methods of treatinga disease, said methods comprising administering to a subject in need oftreatment a heteromultimeric protein of the invention, whereby thedisease is treated. Any of the heteromultimeric proteins of theinvention described herein can be used in therapeutic (or prophylacticor diagnostic) methods described herein.

For example, when the heteromultimeric protein is multivalent, avaluable benefit is the enhanced avidity they pose for their antigen. Inaddition to having intrinsic high affinity on a binding unit (ie, a Fab)to antigen basis, normal IgG antibodies also exploit the avidity effectto increase their association with antigens as a result of theirbivalent binding towards the targets.

A heteromultimeric protein directed against two separate epitopes on thesame antigen molecule may not only provide the benefit of enhancedbinding avidity (because of bivalent binding), but may also acquirenovel properties that are not associated with either of the parentantibodies. Thus, the heteromultimeric proteins of the invention finduse in, for example, the blocking of receptor-ligand interactions.

The heteromultimeric proteins described herein also find use in theapplication of simultaneously blocking the signaling pathways of twotargets with one molecule.

IX. Therapeutic Uses

The heteromultimeric proteins such as antibodies and antibody fragmentsdescribed herein (e.g., an antibody and/or fragment thereof madeaccording to the methods described herein) may be used for therapeuticapplications. For example, such heteromultimeric proteins can be usedfor the treatment of tumors, including pre-cancerous, non-metastatic,metastatic, and cancerous tumors (e.g., early stage cancer), for thetreatment of allergic or inflammatory disorders, or for the treatment ofautoimmune disease, or for the treatment of a subject at risk fordeveloping cancer (for example, breast cancer, colorectal cancer, lungcancer, renal cell carcinoma, glioma, or ovarian cancer), an allergic orinflammatory disorder, or an autoimmune disease.

The term cancer embraces a collection of proliferative disorders,including but not limited to pre-cancerous growths, benign tumors, andmalignant tumors. Benign tumors remain localized at the site of originand do not have the capacity to infiltrate, invade, or metastasize todistant sites. Malignant tumors will invade and damage other tissuesaround them. They can also gain the ability to break off from where theystarted and spread to other parts of the body (metastasize), usuallythrough the bloodstream or through the lymphatic system where the lymphnodes are located. Primary tumors are classified by the type of tissuefrom which they arise; metastatic tumors are classified by the tissuetype from which the cancer cells are derived. Over time, the cells of amalignant tumor become more abnormal and appear less like normal cells.This change in the appearance of cancer cells is called the tumor gradeand cancer cells are described as being well-differentiated,moderately-differentiated, poorly-differentiated, or undifferentiated.Well-differentiated cells are quite normal appearing and resemble thenormal cells from which they originated. Undifferentiated cells arecells that have become so abnormal that it is no longer possible todetermine the origin of the cells.

The tumor can be a solid tumor or a non-solid or soft tissue tumor.Examples of soft tissue tumors include leukemia (e.g., chronicmyelogenous leukemia, acute myelogenous leukemia, adult acutelymphoblastic leukemia, acute myelogenous leukemia, mature B-cell acutelymphoblastic leukemia, chronic lymphocytic leukemia, polymphocyticleukemia, or hairy cell leukemia), or lymphoma (e.g., non-Hodgkin'slymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). A solidtumor includes any cancer of body tissues other than blood, bone marrow,or the lymphatic system. Solid tumors can be further separated intothose of epithelial cell origin and those of non-epithelial cell origin.Examples of epithelial cell solid tumors include tumors of thegastrointestinal tract, colon, breast, prostate, lung, kidney, liver,pancreas, ovary, head and neck, oral cavity, stomach, duodenum, smallintestine, large intestine, anus, gall bladder, labium, nasopharynx,skin, uterus, male genital organ, urinary organs, bladder, and skin.Solid tumors of non-epithelial origin include sarcomas, brain tumors,and bone tumors.

Epithelial cancers generally evolve from a benign tumor to a preinvasivestage (e.g., carcinoma in situ), to a malignant cancer, which haspenetrated the basement membrane and invaded the subepithelial stroma.

Multispecific protein complexes can also be used in these therapeuticapplications, and antibodies that bind HER2 can in particular be used totreat breast cancer, colorectal cancer, lung cancer, renal cellcarcinoma, glioma, or ovarian cancer.

Other subjects that are candidates for receiving compositions of thisinvention have, or are at risk for developing, abnormal proliferation offibrovascular tissue, acne rosacea, acquired immune deficiency syndrome,artery occlusion, atopic keratitis, bacterial ulcers, Bechets disease,blood borne tumors, carotid obstructive disease, choroidalneovascularization, chronic inflammation, chronic retinal detachment,chronic uveitis, chronic vitritis, contact lens overwear, corneal graftrejection, corneal neovascularization, corneal graft neovascularization,Crohn's disease, Eales disease, epidemic keratoconjunctivitis, fungalulcers, Herpes simplex infections, Herpes zoster infections,hyperviscosity syndromes, Kaposi's sarcoma, leukemia, lipiddegeneration, Lyme's disease, marginal keratolysis, Mooren ulcer,Mycobacteria infections other than leprosy, myopia, ocular neovasculardisease, optic pits, Osler-Weber syndrome (Osler-Weber-Rendu),osteoarthritis, Paget's disease, pars planitis, pemphigoid,phylectenulosis, polyarteritis, post-laser complications, protozoaninfections, pseudoxanthoma elasticum, pterygium keratitis sicca, radialkeratotomy, retinal neovascularization, retinopathy of prematurity,retrolental fibroplasias, sarcoid, scleritis, sickle cell anemia,Sogren's syndrome, solid tumors, Stargart's disease, Steven's Johnsondisease, superior limbic keratitis, syphilis, systemic lupus, Terrien'smarginal degeneration, toxoplasmosis, tumors of Ewing sarcoma, tumors ofneuroblastoma, tumors of osteosarcoma, tumors of retinoblastoma, tumorsof rhabdomyosarcoma, ulcerative colitis, vein occlusion, Vitamin Adeficiency, Wegener's sarcoidosis, undesired angiogenesis associatedwith diabetes, parasitic diseases, abnormal wound healing, hypertrophyfollowing surgery, injury or trauma (e.g., acute lung injury/ARDS),inhibition of hair growth, inhibition of ovulation and corpus luteumformation, inhibition of implantation, and inhibition of embryodevelopment in the uterus.

Examples of allergic or inflammatory disorders or autoimmune diseases ordisorders that may be treated using an antibody made according to themethods described herein include, but are not limited to arthritis(rheumatoid arthritis such as acute arthritis, chronic rheumatoidarthritis, gouty arthritis, acute gouty arthritis, chronic inflammatoryarthritis, degenerative arthritis, infectious arthritis, Lyme arthritis,proliferative arthritis, psoriatic arthritis, vertebral arthritis, andjuvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronicaprogrediente, arthritis deformans, polyarthritis chronica primaria,reactive arthritis, and ankylosing spondylitis), inflammatoryhyperproliferative skin diseases, psoriasis such as plaque psoriasis,gutatte psoriasis, pustular psoriasis, and psoriasis of the nails,dermatitis including contact dermatitis, chronic contact dermatitis,allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, colitis such as ulcerative colitis, colitisulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,necrotizing enterocolitis, and transmural colitis, and autoimmuneinflammatory bowel disease), pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, episcleritis), respiratory distresssyndrome, including adult or acute respiratory distress syndrome (ARDS),meningitis, inflammation of all or part of the uvea, iritis,choroiditis, an autoimmune hematological disorder, rheumatoidspondylitis, sudden hearing loss, IgE-mediated diseases such asanaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and/or brainstem encephalitis,uveitis, such as anterior uveitis, acute anterior uveitis, granulomatousuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterioruveitis, or autoimmune uveitis, glomerulonephritis (GN) with and withoutnephrotic syndrome such as chronic or acute glomerulonephritis such asprimary GN, immune-mediated GN, membranous GN (membranous nephropathy),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, allergic reaction, eczemaincluding allergic or atopic eczema, asthma such as asthma bronchiale,bronchial asthma, and auto-immune asthma, conditions involvinginfiltration of T-cells and chronic inflammatory responses, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, systemic lupus erythematosus (SLE) or systemiclupus erythematodes such as cutaneous SLE, subacute cutaneous lupuserythematosus, neonatal lupus syndrome (NLE), lupus erythematosusdisseminatus, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I)diabetes mellitus, including pediatric insulin-dependent diabetesmellitus (IDDM), adult onset diabetes mellitus (Type II diabetes),autoimmune diabetes, idiopathic diabetes insipidus, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includinglymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis,vasculitides, including vasculitis (including large vessel vasculitis(including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), microscopic polyarteritis, CNS vasculitis,necrotizing, cutaneous, or hypersensitivity vasculitis, systemicnecrotizing vasculitis, and ANCA-associated vasculitis, such asChurg-Strauss vasculitis or syndrome (CSS)), temporal arteritis,aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia,Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiaperniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA),Factor VIII deficiency, hemophilia A, autoimmune neutropenia,pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNSinflammatory disorders, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet's orBehcet's disease, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, immune complexnephritis, antibody-mediated nephritis, neuromyelitis optica,polyneuropathies, chronic neuropathy such as IgM polyneuropathies orIgM-mediated neuropathy, thrombocytopenia (as developed by myocardialinfarction patients, for example), including thrombotic thrombocytopenicpurpura (TTP) and autoimmune or immune-mediated thrombocytopenia such asidiopathic thrombocytopenic purpura (ITP) including chronic or acuteITP, autoimmune disease of the testis and ovary including autoimuneorchitis and oophoritis, primary hypothyroidism, hypoparathyroidism,autoimmune endocrine diseases including thyroiditis such as autoimmunethyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto'sthyroiditis), or subacute thyroiditis, autoimmune thyroid disease,idiopathic hypothyroidism, Grave's disease, polyglandular syndromes suchas autoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis,bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barrësyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, primary biliary cirrhosis,pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AIED), autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal garnmopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal segmental glomerulosclerosis (FSGS),endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases,diabetic nephropathy, Dressler's syndrome, alopecia areata, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis,cystic fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis,Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection,echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirusinfection, rubella virus infection, post-vaccination syndromes,congenital rubella infection, Epstein-Barr virus infection, mumps,Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrineophthamopathy, chronic hypersensitivity pneumonitis,keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathicnephritic syndrome, minimal change nephropathy, benign familial andischemia-reperfusion injury, retinal autoimmunity, joint inflammation,bronchitis, chronic obstructive airway disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antobodies, non-malignant thymoma,vitiligo, SCID and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLeishmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T-cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune disorders associated with collagendisease, rheumatism, neurological disease, ischemic re-perfusiondisorder, reduction in blood pressure response, vascular dysfunction,antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,reperfusion injury of myocardial or other tissues, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, acute serious inflammation, chronicintractable inflammation, pyelitis, pneumonocirrhosis, diabeticretinopathy, diabetic large-artery disorder, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

In addition to therapeutic uses, the antibodies of the invention can beused for other purposes, including diagnostic methods, such asdiagnostic methods for the diseases and conditions described herein.

X. Dosages, Formulations, and Duration

The proteins of this invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual subject, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the proteins to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a particular disorder (forexample, a cancer, allergic or inflammatory disorder, or autoimmunedisorder). The proteins need not be, but are optionally, formulated withone or more agents currently used to prevent or treat the disorder. Theeffective amount of such other agents depends on the amount of proteinspresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages. Generally, alleviation ortreatment of a cancer involves the lessening of one or more symptoms ormedical problems associated with the cancer. The therapeuticallyeffective amount of the drug can accomplish one or a combination of thefollowing: reduce (by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100% or more) the number of cancer cells; reduce or inhibit thetumor size or tumor burden; inhibit (i.e., to decrease to some extentand/or stop) cancer cell infiltration into peripheral organs; reducehormonal secretion in the case of adenomas; reduce vessel density;inhibit tumor metastasis; reduce or inhibit tumor growth; and/or relieveto some extent one or more of the symptoms associated with the cancer.In some embodiments, the proteins are used to prevent the occurrence orreoccurrence of cancer or an autoimmune disorder in the subject.

In one embodiment, the present invention can be used for increasing theduration of survival of a human subject susceptible to or diagnosed witha cancer or autoimmune disorder. Duration of survival is defined as thetime from first administration of the drug to death. Duration ofsurvival can also be measured by stratified hazard ratio (HR) of thetreatment group versus control group, which represents the risk of deathfor a subject during the treatment.

In yet another embodiment, the treatment of the present inventionsignificantly increases response rate in a group of human subjectssusceptible to or diagnosed with a cancer who are treated with variousanti-cancer therapies. Response rate is defined as the percentage oftreated subjects who responded to the treatment. In one aspect, thecombination treatment of the invention using proteins of this inventionand surgery, radiation therapy, or one or more chemotherapeutic agentssignificantly increases response rate in the treated subject groupcompared to the group treated with surgery, radiation therapy, orchemotherapy alone, the increase having a Chi-square p-value of lessthan 0.005. Additional measurements of therapeutic efficacy in thetreatment of cancers are described in U.S. Patent ApplicationPublication No. 20050186208.

Therapeutic formulations are prepared using standard methods known inthe art by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences (20^(th) edition), ed.A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).Acceptable carriers, include saline, or buffers such as phosphate,citrate and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone, amino acids such as glycine,glutamine, asparagines, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™, PLURONICS™, or PEG.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations. Optionally, the formulations of theinvention can contain a pharmaceutically acceptable preservative. Insome embodiments the preservative concentration ranges from 0.1 to 2.0%,typically v/v. Suitable preservatives include those known in thepharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben,and propylparaben are preferred preservatives. Optionally, theformulations of the invention can include a pharmaceutically acceptablesurfactant at a concentration of 0.005 to 0.02%.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the heteromultimeric protein, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated heteromultimeric protein(s) remain in thebody for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS-S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The proteins described herein (e.g., a heteromultimeric protein such asa multispecific antibody made according to the methods described herein)are administered to a human subject, in accord with known methods, suchas intravenous administration as a bolus or by continuous infusion overa period of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Local administration may be particularlydesired if extensive side effects or toxicity is associated withantagonism to the target molecule recognized by the proteins. An ex vivostrategy can also be used for therapeutic applications. Ex vivostrategies involve transfecting or transducing cells obtained from thesubject with a polynucleotide encoding a protein of this invention. Thetransfected or transduced cells are then returned to the subject. Thecells can be any of a wide range of types including, without limitation,hemopoietic cells (e.g., bone marrow cells, macrophages, monocytes,dendritic cells, T cells, or B cells), fibroblasts, epithelial cells,endothelial cells, keratinocytes, or muscle cells.

In one example, the protein complex is (e.g., a heteromultimeric proteinsuch as a multispecific antibody made according to the methods describedherein) is administered locally, e.g., by direct injections, when thedisorder or location of the tumor permits, and the injections can berepeated periodically. The protein complex can also be deliveredsystemically to the subject or directly to the tumor cells, e.g., to atumor or a tumor bed following surgical excision of the tumor, in orderto prevent or reduce local recurrence or metastasis.

XI. Articles of Manufacture

Another embodiment of the invention is an article of manufacturecontaining one or more protein complexes described herein, and materialsuseful for the treatment or diagnosis of a disorder (for example, anautoimmune disease or cancer). The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition that iseffective for treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is a heteromultimeric protein (e.g.,an antibody or antibody fragment) of the invention. The label or packageinsert indicates that the composition is used for treating theparticular condition. The label or package insert will further compriseinstructions for administering the heteromultimeric protein compositionto the subject. Articles of manufacture and kits comprisingcombinatorial therapies described herein are also contemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In certainembodiments, the package insert indicates that the composition is usedfor treating breast cancer, colorectal cancer, lung cancer, renal cellcarcinoma, glioma, or ovarian cancer.

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials considered from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forpurification or immunoprecipitation of an antigen (e.g., HER2 or EGFR)from cells. For isolation and purification of an antigen (e.g., HER2 orEGFR) the kit can contain a heteromultimeric protein (e.g., an EGFR/HER2antibody) coupled to beads (e.g., sepharose beads). Kits can be providedwhich contain the heteromultimeric protein(s) for detection andquantitation of the antigen in vitro, e.g., in an ELISA or a Westernblot. As with the article of manufacture, the kit comprises a containerand a label or package insert on or associated with the container. Thecontainer holds a composition comprising at least one heteromultimericprotein (e.g., multispecific antibody or antibody fragment) of theinvention. Additional containers may be included that contain, e.g.,diluents and buffers or control antibodies. The label or package insertmay provide a description of the composition as well as instructions forthe intended in vitro or diagnostic use.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The followingExamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds); ADCC (antibody-dependent cellular cytotoxicity)); BsAb(bispecific antibody); C_(L) (constant domain of light chain); C_(H)(constant domain of heavy chain); CMC (complement-mediatedcytotoxicity); Fab (antigen binding fragment); Fc (crystallizedfragment); Fv (variable fragment (V_(L)+V_(H))); EGFR (epidermal growthfactor receptor); HC (heavy chain); IGFR (insulin-like growth factorreceptor); LC (light chain); scFv (singlechain variable fragment (V_(L)and V_(H) tethered by an amino acid linker); VEGF (vascular endothelialgrowth factor); VEGFR2 (vascular endothelial growth factor receptor 2);V_(H) (variable heavy domain); V_(L) (variable light domain).

EXAMPLES

The present invention is described in further detain in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein. The following examples are offered toillustrate, but not to limit the claimed invention.

Example 1 Construction of Expression Vectors

This example illustrates the nucleic acid construct used to transformhost cells.

Generally, both the heavy and light chain DNA coding sequences werecloned into an expression plasmid that contained separate promoterelements for each of the sequences and antibiotic resistance forselection of bacterial cells that contain the expression plasmid. Thevector constructs also encode the heat-stable enterotoxin II (STII)secretion signal (Picken et al., 1983, Infect. Immun. 42:269-275, andLee et al., 1983, Infect. Immun. 42:264-268) for the export of theantibody polypeptides into the periplasmic space of the bacterial cell.Transcription of each chain is controlled by the phoA promoter (Kikuchiet al., 1981, Nucleic Acids Res., 9:5671-5678) and translational controlis provided by previously described STII signal sequence variants ofmeasured relative translational strength, which contain silent codonchanges in the translation initiation region (TIR) (Simmons and Yansura,1996, Nature Biotechnol. 14:629-634 and Simmons et al., 2002, J. ImmunolMethods, 263:133-147). A schematic drawing of the knob and hole plasmidsis shown in FIG. 2A and FIG. 2B, respectively.

While the present invention does not rely on specific antibody bindingsequences, and is applicable to any half-antibody combinations, theExamples herein are directed to heteromultimeric antibodies directed toc-met, EGFR, IL-4 and IL-13. Examples of anti-c-met antibodies are givenin U.S. Pat. No. 7,472,724, and U.S. Pat. No. 7,498,420. Examples ofanti-EGFR antibodies are given in U.S. Provisional Application61/210,562 (filed 20 Mar. 2009), US Pat. Appln. Pub. No. 20080274114(published 6 Nov. 2008) and U.S. Pat. No. 5,844,093 (granted 1 Dec.1998). Examples of anti-IL-13 antibodies are described in U.S. Pat. No.7,501,121 (granted 10 Mar. 2009), U.S. Pat. No. 7,615,213 (granted 10Nov. 2009), WO 2006/085938 (published 17 Aug. 2006), US Pat Appln. Pub.No. 20090214523 (published 27 Aug. 2009), and U.S. Pat. No. 7,674,459(granted 9 Mar. 2010). Examples of anti-IL-4 antibodies are described inUS Pat. Appln. Pub. No. US 20080241160 (published 2 Oct. 2008), and U.S.Pat. No. 6,358,509 (granted 19 Mar. 2002).

Each half-antibody had either a knob (protuberance) or a hole (cavity)engineered into the heavy chain as described in U.S. Pat. No. 7,642,228.Briefly, a C_(H)3 knob mutant was generated first. A library of C_(H)3hole mutants was then created by randomizing residues 366, 368 and 407that are in proximity to the knob on the partner C_(H)3 domain. In thefollowing examples, the knob mutation is T366W, and the hole hasmutations T366S, L368A and Y407V in an IgG1 backbone. Equivalentmutations in other immunoglobulin isotypes is easily determined by oneskilled in the art. Further, the skilled artisan will readily appreciatethat it is preferred that the two half-antibodies used for thebispecific be the same isotype. Half-antibodies of different isotypesmay be used but may need further mutations.

Although the vector described in this Example is for either theanti-c-Met or anti-EGFR half-antibody, one skilled in the art willreadily appreciate that any antibody can be encoded in the plasmid. Thestarting plasmid for all constructs used herein is the previouslydescribed anti-tissue factor separate cistron plasmid, paTF50, withrelative TIRs of 1 for heavy and 1 for light (Simmons et al., 2002, J.Immunol Methods, 263:133-147, and U.S. Pat. No. 6,979,556). An increasein the relative TIR strengths was used to increase the expression titersof these half-antibodies.

Example 2 Heteromultimeric Protein Production Using Separate CellCultures

The following example shows the production of heteromultimeric proteinswhen the cells expressing the monomeric components are grown in separatecultures. In this method the cells are grown and induced to express thehalf-antibody in separate cultures. In one method, the host cellcultures may be combined before protein purification. In another methodthe components may be purified first and then combined to form theheteromultimeric protein.

In both methods, a nucleic acid encoding the first hinge-containingpolypeptide (e.g., a half-antibody (knob)) is introduced into a firsthost cell and a nucleic acid encoding the second hinge-containingpolypeptide (e.g., a half-antibody (hole)) is introduced into a secondhost cell. Although this example illustrates the formation of a BsAb oneskilled in the art will readily appreciate that the methods describedare applicable to any heteromultimeric protein comprising a hingeregion, e.g., affibodies, etc.

Method #1—Independent Production of Knob Half-Antibody and HoleHalf-Antibody in Separate Cultures, Separate Purification of theHalf-Antibodies, Mixing and Redox to Form Intact BsAb.

Half-antibodies containing either the knob or hole mutations weregenerated in separate cultures by expressing the heavy and light chainsusing the constructs described in Example 1 in a bacterial host cell,e.g., E. coli. See FIG. 3B and FIG. 4A. In this Method #1 the knobhalf-antibody was an anti-EGFR and the hole half-antibody was ananti-c-met. The expression plasmids of Example 1 were introduced into E.coli host strains 33D3 (Ridgway et al. (1999) 59 (11): 2718) or 64B4(W3110 ΔfhuA ΔphoA iivG+Δprc spr43H1 ΔdegP ΔmanA lacl^(q) ΔompT) andtransformants were selected on carbenicillin containing LB plates.Transformants were then used to inoculate an LB starter culturecontaining carbenicillin, and this was grown overnight with shaking at30° C. The starter culture was diluted 100× into a phosphate limitingmedia C.R.A.P. (Simmons et al., 2002, J. Immunol Methods, 263:133-147)containing carbenicillin, and this was grown for 24 hours with shakingat 30° C. The cultures were centrifuged, and the cell pellets frozenuntil the start of antibody purification. The pellets were thawed andresuspended in an extraction buffer containing 25 mM Tris-base adjustedto pH 7.5 with hydrochloric acid, 125 mM NaCl and 5 mM EDTA (TEB or TrisExtraction Buffer) with a volume to weight ratio of 100 mL TEB per 5grams of cell pellet, and extracted by disrupting the cells usingmicrofluidics by passing the resuspended mixture through a MicrofluidicsCorporation model 110F microfluidizer (Newton, Mass.) three times. Thebacterial cell extract was then clarified by centrifugation for 20minutes at 15,000λg and the supernatant collected and filtered through a0.22 micron acetate filter prior to purification.

Each half-antibody was purified separately by Protein A capture followedby cation exchange chromatography. Clarified cell extracts from the knobhalf-antibody were loaded onto a 1 mL HiTrap MabSelect SURE column fromGE Healthcare (Pistcataway, N.J.) at 2 mL/min. After loading the columnwas washed with 10 column volumes (CV) of 40 mM sodium citrate, pH 6.0,125 mM sodium chloride, and 5 mM EDTA followed by 5 column volumes of 20mM sodium citrate at pH 6.0 to facilitate capture by the cation exchangecolumn. The affinity captured half-antibodies were eluted with 10 columnvolumes (CV) of 0.2 mM acetic acid (pH 2-3) and directly captured on a 1mL HiTrap SP-HP strong cation exchange column from GE Healthcare. Thecolumn was washed with 10 CV of buffer A containing 25 mM2-(N-morpholino)ethanesulfonic acid (MES) pH 5.8. The half-antibodieswere eluted with a linear gradient of 0-50% buffer B (25 mM MES, pH 5.8and 1 M sodium chloride (NaCl)). Both proteins eluted between 20-40% Band the eluant peak as determined by UV absorbance at 280 nm and bynon-reducing SDS-PAGE analysis of the collected fractions were pooledseparately as the knob or hole half-antibody. Both proteins generallyexhibited a major elution peak and all fractions that contained heavychain and light chain species that were oxidized to one another wereincluded in the pool. Analysis of the purified half-antibodies byreducing and non-reducing SDS-PAGE are shown in FIG. 4B. The resultsindicate that most of the expressed and captured protein is 75 kD insize. We confirmed this by ESI-TOF mass spectrometry shown in FIG. 4C.The mass of the half-antibodies were the expected masses indicating thatthere were no disulfide adducts on any cysteine, including the twocysteine residues in the hinge region. To determine if the hingecysteines were reduced exhibiting a reactive free thiol, the proteinswere reacted in at a neutral pH with 1 mM N-ethylmaleimide (NEM) for onehour before analysis by mass spectrometry. The mass of the protein wasunchanged indicating that the hinge cysteines were oxidized to eachother most likely in an intrachain disulfide, e.g., a cyclic disulfide.In order to assemble a fully intact, bispecific antibody using these twohalf-antibodies (knob and hole), it was necessary to first reduce theintrachain disulfides at the hinge region to liberate the cysteine freethiols so that they could subsequently be oxidized to the other heavychain to form the 150 kD bispecific antibody.

To accomplish the annealing, reduction and reoxidation of the twocomplementary half-antibodies to form the intact bispecific moleculesthe following procedure was developed. After independent isolation, thepurified proteins were combined together at equal mass in the Pool stepof the procedure (shown in FIG. 5A), the pH of the pool was adjusted to7.5 by adding one-tenth volume of 1 M Tris, pH 7.5, and proteins werereduced with 0.5 mM Tris[2-carboxyethyl] phosphine (TCEP) at roomtemperature. After reduction for 2 hours the pooled proteins were bufferexchanged into 25 mM Tris, pH 7.5, and 125 mM NaCl using 5 mL ZebaDesalt spin columns (Pierce, Rockford, Ill.) resulting in a volume ofabout 4 mLs of a protein concentration of 1 mg/mL. The proteins werethen annealed by heating the mixture to 52° C. for 25 minutes followedby cooling to room temperature, about 20° C. The annealed antibodieswere concentrated using 10 kD MW cutoff spin concentrators to a volumeof 0.5 mL with a protein concentration of about 8 mg/mL and oxidized bythe addition of 300 micromolar dehydroascorbic acid (DHAA) to thereaction mixture from a stock solution of 100 mM DHAA dissolved indimethylsulfoxide. The amount of DHAA added for oxidation is about10-fold excess over the protein molar concentration. After oxidationovernight at room temperature, the oxidized material was run on an S-200gel filtration column (22 mL S200 Tricorn from GE Healthcare) in abuffer containing 25 mM MES pH 6.0 and 300 mM NaCl. The intact antibodywas pooled and diluted 10-fold in water. The BsAb protein was thenpurified by weak cation exchange chromatography using a carboxymethyl(CM) resin (1 mL HiTrap CM-FF, GE Healthcare) with a pH gradient elutionfrom 4.5 to 9.2. The buffer A and B composition consisted of 20 mMsodium citrate, 30 mM MES, 20 mM HEPES, 20 mM imidizole, 20 mM Tris, 20mM CAPS, and 25 mM NaCl, where the A buffer is adjusted to pH 4.2 withHCl and the B buffer is adjusted to pH 9.2 (or 10.4) using NaOH. Thepurified material obtained after CM chromatography was analyzed by massspectrometry to determine the exact molecular composition (FIG. 4D).Mass spec analysis indicated that the only detectable intact antibodyproduct was with a MW of 146,051.89, which matches nearly identicallywith the heterodimeric knob-hole species anti-EGFR/anti-c-met with atheoretical MW of 145,051.75. The yield of this procedure, beginningwith about 2 mg of the knob and 2 mg of the hole was about 0.5-1 mg.

For large scale production of antibodies for in vivo experimentationsuch as the determination of pharmacokinetic properties in non-humanprimates, 100 mg to gram scale quantities of antibody are needed. Wedeveloped a procedure using a separate, independent culture for eachhalf-antibody as shown in FIG. 5A to produce intact bispecificantibodies in these quantities. For these preparations, 10 literfermentations were required to produce cell pellets or whole broth withsufficient quantities of antibody (Simmons et al., 2002. J. Immunol.Methods, 263:133-147, and U.S. Pat. No. 6,979,556). In the course ofexperimentation either cell pellets or bacterial whole broth were usedfor biomass containing expressed half-antibodies. In some cases, asignificant fraction of the antibody had leaked out into the media,where whole broth gave higher yields. For cell pellets, the material wasresuspended in extraction buffer containing 25 mM Tris, pH 7.5, 5 mMEDTA, and 125 mM NaCl and lysed by microfluidization using a ModelHC80003A microfluidizer from Microfluidics (Newton, Mass.). Whole brothwas directly microfluidized without the addition of additives. In bothcases, three passes of the material through the instrument was done. Inthis example, we prepared 500 mg of two versions of a bispecificantibody targeting the cytokines interlukin-4 (knob) and interleuikin-13(hole).

The first version of the bispecific contained a human IgG1a Fc with onlythe knob and hole mutations and the second contained a further modifiedFc with two mutations, T307Q and N434A, that lead to a greater affinityfor the neonatal Fc receptor (FcRn). The second versions are expected toimpart a slower clearance and longer half-life for the antibody. Thehole antibody (targeting IL-4) and the knob antibody (targeting IL-13)of both versions of the Fc (WT-Fc for the former and FcRn-variant forthe later) were both grown separately in 10 liter fermentation and thewhole broth containing growth media and bacterial cells were homogenizedand purified independently. After microfluidization of the whole broth,the extract was treated with an equal volume of 0.4% polyethyleneimine(PEI) (pH 9.0) to prepare the extract for clarification bycentrifugation. The mixture was stirred for 3 hours at room temperatureor overnight at 4° C. PEI caused extensive precipitation of the extractwhich was clarified by centrifugation at 15,000×g for 45 minutes. Thesupernatant was subsequently filtered by 0.22 micron filters beforeloading on a 100 mL Mab Select SURE Protein A capture column. Theextract was loaded at 20 ml/min and washed with 40 mM sodium citrate, pH6.0, and 100 mM NaCl until the UV absorbance at 280 reached a stablebaseline, generally about 10 column volumes (CV). The wash buffer waschanged to 20 mM sodium citrate, pH 6.0 and washed for about 2 CV. Thecaptured half-antibody was eluted using 0.2 M acetic acid. Afterisolation by Protein A the antibodies were purified by cation exchangechromatography using S-FF resin (GE Healthcare) or gel filtrationchromatography using S200 resin (GE Healthcare) to remove impurities andaggregates. The purified half-antibodies were mostly the ˜75 kD speciesas seen in FIG. 5B. After the second isolation step, 500 mg of eachhalf-antibody were pooled together at a concentration of 1 mg/mL and thepH was adjusted to 7.5 using 1 M Tris, pH 7.5. The mixture was heated to37° C. in an incubator and monitored by gel filtration for the emergenceof the 150 kD antibody species. After 2 hours, the annealing wascomplete showing complete conversion to the dimeric 150 kD species andthe mixture was cooled to room temperature. The proteins were reduced bythe addition of 2 mM DTT for two hours at 24° C. and subsequentlyconcentrated to 20 mg/mL using 10 kD cutoff spin filters. Theconcentrated solution was oxidized by dialysis overnight in a buffercontaining only 25 mM Tris, pH 8.0. The oxidized material wassubsequently analyzed for purity and aggregation. The intact antibodyspecies was determined by mass spectrometry to be the intact, fullyoxidized heterodimeric bispecific molecule however gel filtration andSDS-PAGE analysis indicated the presence of significant amounts ofaggregate, some of which was clearly the result of disulfide linkedmultimers (DATA not shown). To further purify the bispecific antibodyfor in vivo experimentation, the antibody was separated over an S-200gel filtration column in Tris, pH 7.5 and 125 mM NaCl. The purifiedmaterial exhibited a greater than 30% loss of material due to theremoval of introduced aggregates. For the final stages of thepreparation, the protein was adhered to a cation exchange column, washedwith 0.1% TX114 in 50 mM sodium acetate, pH 5.0, to remove contaminatingendotoxin, and eluted with a high pH buffer containing 50 mM Tris, pH8.0. The eluted protein was then formulated by dialysis into a buffersuitable for in vivo experimentation and stored at 4° C. The finalmaterial consisting of the WT-Fc and the FcRn-variant was analyzed bySDS-PAGE, mass spectrometry, LAL assays for determining contaminatingendotoxin levels, and gel filtration analysis. The results of theSDS-PAGE are shown in FIG. 5C, and indicate that the major species isthe intact bispecific antibody at 150 kD. FIG. 6A shows the biologicalactivity of the antibodies in a TF-2 cell proliferation assay testingneutralization of the cytokines IL-4 and IL-13. For the assay,anti-IL-4/IL-13 bispecific, anti-IL-4 and anti-IL-13 antibodies wereused at a starting concentration of 25 ug/ml and serially diluted 10fold in a 96 well culture plate (Falcon, Cat #353072) to a finalconcentration of 0.025 pg/ml in assay media (culture media withoutrhGM-CSF) or assay media containing 0.4 ng/ml human IL-4 (R&D Systems,Catalog #204-IL) plus 20 ng/ml human IL-13 (Genentech Inc.) in a finalvolume of 50 ul/well. Diluted antibodies were pre-incubated for 30minutes at 37° C.

Following preincubation, TF-1 cells cultured in RPMI 1640 (Genentech,Inc.) 10% Fetal Bovine Serum (HyClone, Cat #SH300071.03), 2 mML-glutamine 100 units/mL Penicillin 100 μg/mL Streptomycin (Gibco, Cat#10378) and 2 ng/mL rhGM-CSF (R&D Systems, Cat #215-GM) were washed 2times with assay media and resuspended in assay media to obtain a finalconcentration of 2×10⁵ cells/ml. 50 ul of cells were added to each wellcontaining either the diluted antibodies, assay media plus IL-4 andIL-13 cytokines (maximal proliferation control) or assay media alone(background control). All samples were plated in duplicate. Plates wereincubated at 37° C. at 5% CO₂ for 4 days. 1 uCi ³H Thymidine (PerkinElmer, Cat #NET027005MC) was added to each well during the final 4 hrsof incubation. Plates were harvested onto a Unifilter-96 GF/C (PerkinElmer, Cat #6005174) using a Packard Filtermate, ³H thymidineincorporation was measured using a TopCount NXT (Perkin Elmer). Data wasplotted using KaleidaGraph. The results indicate that the WTanti-II-4/anti-IL-13 bispecific antibody is as effective as IgG antibodycombinations of IL-4 and IL-13 in neutralizing IL-4 and IL-13 activity.

The two antibodies (WT anti-IL-4/anti-IL-13 and FcRn-variantanti-IL-4/anti-IL-13) were then tested for their pharmacokinetic (PK)properties in cynomologous monkey. Using a single dose injection, the WTmolecule formulated in 20 mM histidine-acetate, pH 5.5, 240 mM sucrose,and 0.02% Tween 20 at 10.8 mg/mL and 1 mg/mL and the FcRn-variant in 20mM sodium phosphate, pH 7.5, 240 mM sucrose, and 0.02% Tween 20 at 10.5mg/mL, were administered by IV injection. The dosing level was 20 mg/kgand 2 mg/kg for the two WT concentrations and 20 mg/kg for theFcRn-variant. Serum samples from two female and two male monkeys thatwere injected with the three treatments were taken periodically over thecourse of 42 days. The serum samples were assayed for the intactbispecific antibody by ELISA wherein one antigen, either IL-4 or IL-13,was coated onto the plates and the antibody subsequently captured fromthe serum. The amount of captured bispecific antibody present wasdetermined by detection with a second biotinylated ligand either IL-13or IL-4 (whichever ligand had not been coated onto the plates), andenzyme-coupled streptavidin. The results in FIG. 6B shows the expectedtwo compartment clearances of the three samples. The PK properties ofthe two different versions of the antibody are shown in Table 2 incomparison to two other antibodies that are derived from CHO productionhosts (Avastin and Herceptin) and contain Fc-glycosylation. It is clearthat the E. coli produced bispecific antibody is similar to the CHOderived antibodies from a standard process and that the FcRn-variant hasa longer half-life.

TABLE 2 Population Mean Vc CL T½ (% RSE) (mL/kg) (mL/kg/day) (day) WT29.0 (9.48) 4.49 (7.66) ~10 FcRn 15.8 (5.72) 2.11 (2.47) ~18 Avastin 4.3~12 Herceptin 5.5 ~9Method #2—Production of Knob Half-Antibody and Hole Half-Antibody inSeparate (i.e., Independent) Cultures, Mixing Whole Broth Prior toPurification of the Half-Antibodies and Lysis without the Addition of aReductant to Form Intact BsAb.

This method was an attempt to reduce the number of steps in the processby purifying the knob and the hole half-antibodies at the same time.Therefore, fermentation broths were mixed prior to pelleting andresuspending in extraction buffer. It was thought that each host cellwould release its expressed half-antibody containing the cyclicdisulfide within the hinge region into the extraction buffer upon cellmembrane disruption. Subsequently, the purification of bothhalf-antibodies could be done simultaneously followed by theredox-annealing step to form the intact BsAb. Surprisingly, wediscovered that the knob-hole antibodies heterodimerized and oxidized ontheir own to form a full length antibody (˜150 kD) at greater than 20%of the combined total of the intact and half-antibody (˜75 kD) (seeTable 3).

The knob half-antibody and hole half-antibody expressing host cells weregrown and induced in separate cultures using the process as described inMethod #1, supra. The whole cell fermentation broth from each culturewas mixed with the other at three different volume ratios and thencentrifuged to form a single cell pellet. The whole cell fermentationbroths were mixed together to a final volume of 500 mL at an(anti-c-met):(anti-EGFR) ratio of 1:1, 2:1 or 1:2, with the intent tomatch recovery of the two antibodies in relatively equal abundance andknowing that the anti-EGFR half antibody expressed similar to the cMetantibody under the same conditions. Each cell pellet was resuspended inextraction buffer and lysed. Protein was extracted and purified byProtein-A chromatography followed by cation exchange chromatography asdescribed in Example 2, Method #1. The extraction buffer contained 25 mMTris, pH 7.5, 125 mM NaCl, and 5 mM EDTA. When purified separately, eachof the knob-half-antibody and hole-half-antibody form a cyclic disulfidewithin the hinge region, i.e., an intrachain disulfide, preventingcovalent association of the knob and hole heavy chains. However, it wasfound that when the first and second host cells were lysed togethereither after co-culturing or after mixing whole fermentation brothsprior to centrifugation, there was some level of assembly into theintact antibody species. FIG. 7 shows the intact antibody speciesobserved in the three ratios. This suggested that modifications to theprocedure could result in spontaneous formation of the intact bispecificantibody which could substantially eliminate the need for additionalchemistry steps.

Quantitation of the two protein species was done by separating 5micrograms of protein by SDS-PAGE using a Novex 4-20% Tris-Glycine gel(Invitrogen, Carlsbad Calif.). After electrophoresis the gel was stainedwith colloidal Coomassie stain containing 150 mM ammonium sulfate, 1.74M acetic acid, 10% methanol and 0.4 g/L Coomassie Dye R250 in water. Thegel was destained with 10% acetic acid in water and subsequentlyequilibrated in Gel-Dry Drying Solution (Invitrogen) and dried betweentwo sheets of cellophane. After drying the gel, the protein bands werequantified by the Odyssey IR imaging system (LI-COR Biosciences,Lincoln, Nebr.) at 700 nm.

TABLE 3 Licore fluorescent signals for intact antibodies and half-antibodies after mixed isolations from two separately grown knob andhole cultures. [this is a measure of a hinge] Volume Ratio 150 kD 75 kDCombined % of 150 (c-met:EGFR) RFUs RFUs RFUs RFU/total 1:1 36.01 98.78134.8 26.72 2:1 36.8 107 143.8 25.59 1:2 34.64 107.83 142.5 24.31Method #3—Production of Knob Half-Antibody and Hole Half-Antibody inIndependent Cultures, Independent Centrifugation, Pellets Mixed &Resuspended Followed by Lysis, and Purification of the BsAb without theAddition of a Reductant.

This method is an attempt to reduce the number of steps in the processby purifying the knob and the hole at the same time.

The cells are cultured independently and pelleted by centrifugation. Thepellets are mixed and resuspended together in extraction buffer. It isbelieved that the half-antibodies will be released into the extractionbuffer upon disruption of the cell membranes and that a similar productprofile will be seen as with Method #2, above.

Example 3 Heteromultimeric Protein Production Using a Single Mixed CellCulture

This example illustrates the formation of heteromultimeric proteins froma culture comprising two host cell populations, wherein there is noaddition of a reductant in the process.

Method #4—Production of Knob Half-Antibody and Hole Half-Antibody fromDifferent Cell Populations in the Same Culture to Form Intact BsAbwithout the Addition of Reductant.

Co-culture experiments were first performed in 0.5 liter shake flaskswith two different E. coli transformants containing either a knob orhole half-antibody. For this experiment, a starter culture of both theknob (anti-EGFR) and hole (anti-cMet) half-antibodies were produced byovernight culture in LB-media (100 μg/ml carbenicillin) in 5 mL culturesat 30° C. The overnight cultures of equal OD₆₀₀ were used to inoculate500 ml complete CRAP-media (100 μg/ml carbenicillin) in three differentratios (anti-EGFR:anti-cMet; 1.5:1, 1:1 and 1:1.5) keeping the totalseed volume to 1/100 of the culture. Cells were grown for 24 hrs at 30°C., 200 rpm. The cells were then pelleted by centrifugation (6750×g, 10minutes, 4° C.) and used for purification.

The cells were resuspended in extraction buffer containing 25 mM Tris,pH 7.5, 5 mM EDTA, and 125 mM NaCl at a ratio of 100 mL per 10 g cellpellet. After extraction by microfluidization and preparation forchromatography as described in Example 2, the cell extracts of the threedifferent ratios were purified by first capturing the bispecificantibody on a Mab Select SURE 1 mL HiTrap column (GE Healthcare, S. SanFrancisco, Calif.) and with a column wash buffer containing only 40 mMsodium citrate at pH 6.0. After washing and elution as described inExample 2, the protein A capture pools were loaded onto an SP-HP cationexchange column and purified as described in Example 2. After separationby cation exchange, the chromatographic peaks from each of the threepurifications were pooled and concentrated to a volume of about 50-100microliters, and with a protein concentration of about 15 mg/mL. Theinitial inoculation ratios appeared to make a difference in the finalamount of intact antibody, and this was a higher proportion of intactbispecific antibody to lower molecular weight forms than was observedwhen the cell pellets were mixed together after overnight culturing at37° C. See Table 4.

TABLE 4 Inoculation 150 kD 75 kD Combined % of 150 Ratio RFUs RFUs RFUsRFU/total 1.5 to 1  11.71 10.28 22.0 53.25 1 to 1 9.09 8.96 18.1 50.36 1 to 1.5 7.28 8.71 16.0 45.53

To determine if co-culture can be extended to the 10 liter fermentationscale, which is critical for scale up procedures, several experimentswere done with the anti-EGFR and anti-cMet half-antibodies. For 10 literfermentations, an inoculation starting culture was used that contained a1:1 cell ratio of anti-EGFR and anti-cMet. The 10 liter co-cultures weregrown under identical conditions as for the single half-antibodycultures described in Example 2. Either cell pellet or whole broth wasused for extraction and isolation of the antibody material, also asdescribed above. For extraction of material from the cell pellets, about2.5 kg of paste was produced from one 10 liter fermentation. The cellpellets were resuspended in 5 Liters of buffer containing 25 mM Tris, pH7.5, and 125 mM NaCl. The pellet was treated with a polytron mixer for 2minutes prior to resuspending the pellet, and then microfluidized,clarified, and prepared for Protein A capture as described in Example 2.The fermentation experiment was repeated two more times and the resultsof the co-culture isolation from 10 liter fermentors are shown in FIG.8C. Mass spectrometry was used to characterize the ˜150 kD protein andthe ˜75 kD protein to determine the molecular components. To oursurprise, the dominant upper MW protein is the bispecific antibody andthe ˜75 kD protein was primarily the cMet half-antibody due to itsdifferential expression profile. This indicates that the bispecificantibody has completely formed without the need for additional chemistrysteps. Because the bispecific antibody is a 1:1 stoichiometriccombination of the knob and hole half-antibodies, the presence of only a75 kD protein indicates that the majority of the limiting half-antibodyhad been spontaneously incorporated into the intact bispecific antibody.

This observation led to the development of a simplified expression andpurification scheme as shown in FIG. 8D. After protein A capture, theantibody was diluted 1:1 with a buffer containing 1.5 M ammonium sulfateand 25 mM sodium phosphate pH 6.5 and loaded onto a hydrophobicinteraction column (HIC) Dionex Pro Pac HIC-10 4.6 mm×100 mm (Sunnyvale,Calif.). A gradient of 30-60% B, with the A buffer composed of 25 mMsodium phosphate, pH 6.95, and 1.5 M ammonium sulfate, and the B buffercomposed of 25 mM sodium phosphate, pH 6.95, and 25% isopropyl alcohol.Proteins were separated with a 15 CV gradient. The protein separatedinto two major species, one containing the intact bispecific antibodyand the other containing the excess anti-EGFR half-antibody. The resultsof the chromatographic separation are shown in FIG. 8E. The fractionscontaining the intact antibody were pooled and treated to remove anyremaining contaminating endotoxin by adherence to an S-FF column in a 25mM sodium acetate buffer at pH 5.0, washing with the same acetate buffercontaining 0.1% Triton X114, and then removing the detergent by washingwith the starting acetate buffer. The protein was eluted from the S-FFcolumn using 25 mM Tris, pH 8.0, pooled, and analyzed by SDS-PAGE, massspectrometry and LAL assays for endotoxin. The protein contained 0.076EU/mg of endotoxin in the final preparation, indicating that it issuitable for in vivo applications. The final characterization is shownin FIG. 8F. The SDS-PAGE analysis shows a majority of the protein to bethe final intact bispecific antibody, and the mass spec analysis showsthe expected molecular weight for the bispecific antibody, and the lackof any contaminating species, in particular the homodimeric forms thatcould be present. The comparison of the modified procedure usingcoculturing compared to the procedure that requires annealing and redoxchemistry is shown in FIG. 8G.

Method #5—Production of Knob Half-Antibody and Hole Half-Antibody in theSame Culture to Form Intact BsAb Using Differing Knob:Hole Ratios.

This example shows that host cells using similar expression constructs(differing only in the half-antibody to be expressed) do not outgroweach other and produce intact BsAb.

Experiments have demonstrated that controlling the ratio of either chainis easily done by adjusting the inoculation ratio prior to expansion andexpression. The two strains do not outgrow one another.

To determine if the ratio of inoculation is preserved over thefermentation of a co-culture, an experiment was conducted to determinethe amount of the knob or hole heavy chain that was present at the endof a 24 hour fermentation of co-cultures with different cell ratios.Cells harboring either the knob (anti-EGFR) or hole (anti-c-Met) plasmidwere grown separately in LB-media (100 μg/ml carbenicillin) over nightat 30° C. The starter culture was used to inoculate complete CRAP-media(100 μg/ml carbenicillin) with different ratios of overnight culturekeeping the combined inoculation volume at 1:100 of the final culture.The ratios tested for anti-EGFR:anti-c-Met were 10:1, 5:1, 2:1, 1:1,1:2, 1:5, and 1:10. After culturing for 24 hrs at 30° C. cell sampleswere obtained and analyzed by non-reduced SDS-PAGE (12% TrisGlycine)followed by Western blotting with Goat anti-Human IgG-Fc Antibody HRPconjugated (Bethyl Laboratories, Inc., Montgomery, Tex.). The heavychains of the two species resolve by SDS-PAGE and the result is shown inFIG. 8B. The amount of each half-antibody correlates with theinoculation ratio of the co-culture, indicating that cells harboringplasmids encoding different half-antibodies do not outgrow each other ina co-culture.

Method #6—Production of Knob Half-Antibody and Hole Half-Antibody in theSame Culture to Form Intact BsAb—Membrane Permeabilization.

This example shows that membrane permeabilization releases thehalf-antibodies into the media and with the subsequent formation of anintact BsAb without the need for additional chemistry (e.g., redox orcoupling).

It is known that mutations leading to the loss of lipoprotein synthesisalters the cell membrane of E. coli conferring leakiness of periplasmicproteins into the media and also renders E. coli hypersensitive to EDTA(Hirota, Y. et al. PNAS 74:1417-1420 (1977)). The release of expressedantibody from strain 65G4 (W3110 AfhuA ΔphoA ilvG+Δprc spr43H1 ΔdegPΔmanA lacl^(q) ΔompT Δlpp) with and without addition of EDTA wascompared. Cells expressing either α-IL-4 (hole) or α-IL-13 (knob) wereco-cultured as described in Method #4 in a 1:1 ratio and grown in anincubator shaker at 200 rpm for 20 hrs at 30° C. At the end of theincubation the culture was split into three equal aliquots. One sampleserved as a control with no EDTA added. To the other two samples EDTA,pH 8.0, was added to 10 mM final concentration. Incubation was continuedfor all samples for 30 minutes, after which one of the EDTA treatedsamples had MgCl2 added to 20 mM final concentration. All samples wereincubated for an additional 30 minutes in the incubator shaker beforeremoving cells by centrifugation (9200×g, 20 minutes, 4° C.) and thesupernatant filtered through a GF/F filter (Whatman, Piscataway, N.J.)and 0.2 μm PES filter (Nalgene, Rochester, N.Y.). DNasel, bovinepancreas (Sigma, St. Louis, Mo.) can be added to 4 mg/l to improvefiltration.

The filtered supernatant was then directly loaded over a 1 mL Protein AMabSelect SURE HiTrap column (GE Healthcare) as described previously.The captured protein was eluted with acetic acid as described above andthe peak recovery of the protein can be seen in FIG. 9A. The resultsshow that the total UV absorbance increases in the EDTA treated samples.This absorbance is intact bispecific antibody and excess half-antibody.See FIG. 9B and FIG. 9C.

In a separate experiment the anti-IL-4 and anti-IL-13 half-antibodieswere expressed separately or as a 1:1 co-culture of 65G4 cells. Cellswere cultured as described above (Method #4) with the exception ofsupplementing the complete CRAP media with Silicone Antifoam (Fluke,Buchs, Switzerland) to 0.02% (v/v). After culturing the cells for 24hrs, 30° C., 200 rpm in an incubator shaker, EDTA, pH 8.0, was added to10 mM final concentration and incubation continued for one hour beforeadding MgCl2 to 20 mM. Cells were harvested by centrifugation (6750×g,10 minutes, 4° C.), the supernatant filtered (0.2μ PES, Nalgene,Rochester, N.Y.) and antibodies were captured by protein A as describedabove and analyzed by SDS-PAGE and mass spectrometry. The results shownin FIG. 9D indicate that intact bispecific antibody formation isobserved only in the presence of both halves of the bispecific.Additionally, the majority of the anti-IL-13 antibody was incorporatedinto the bispecific antibody without any additional redox chemistry asmass spec analysis indicated that the 75 kD protein band was mostly theanti-IL-4 half-antibody. The protein A purified bispecific antibody wasdiluted 1:1 with ammonium sulfate buffer and further purified with a 7.5mm×150 mm ProPac HIC-10 column (Dionex, Sunnyvale, Calif.) using thesame procedure as described in Example 3. The intact bispecific antibodywas found to elute at a retention time of 99.68. This peak was pooledand analyzed by SDS-PAGE in non-reducing conditions and found to benearly entirely composed of the intact antibody species. To confirm thatthis protein was a pure heterodimeric bispecific molecule, we analyzedthe protein by ESI-TOF LC/MS. About 10 micrograms of the bispecificantibody were injected onto a PLRP-S 300 A 3 micrometer 50×2.1 mmreverse phase column (Polymer Laboratories) and separated by a 4.3minute gradient of 34-45% 0.05% TFA and acetonitrile using an Agilent1200 Series HPLC and a flow rate of 0.5 mL/min and a column heater at80° C. Protein eluting from the LC was analyzed by an Agilent 6210 TOF.A single peak containing protein was observed, and this peak wasdeconvoluted using Agilent Mass Hunter software version B.02.00 using amass range of 50,000-160,000, 1.0 Da step, 30.0 S/N threshold, averagemass of 90, an unlimited mass range and an isotope width set toautomatic. The majority of the signal representing the expected mass ofthe bispecific molecule. The mass for the intact heterodimericbispecific calculated from the amino acid sequence is 144,044 which iswithin 1-2 Daltons of the measured mass, whereas the calculated massesof the possible homodimeric proteins are 144,954.6 for anti-IL-4 and145, 133.4 for anti-IL-13.

We tested if different inoculation ratios would again persist throughoutthe culture for this set of antibodies and also in the context of theIpp deletion of 65G4. Seed cultures with either anti-IL-4 (hole) oranti-IL-13 (knob) of equal OD₆₀₀ were used to inoculate 500 ml CRAPmedia at 2:1 and 1:2 ratios, cultured and permeabilized at the end ofthe fermentation as described before (see Method #6). The two differentmedia preparations were purified by Protein A capture followed by HICseparation as described above, except that the pH of the HIC A and Bbuffers were lowered to 6.5. The results of the two different startingculture ratios are shown in FIG. 9E. It is observed that the majority ofthe protein is the intact bispecific antibody. The other peaks werecharacterized by mass spectrometry and labeled on the FIG. 9E. Theanti-IL-13 half-antibody is slightly detected, and a significant amountmore of anti-IL-4 is seen. In the 33/66 ratio of anti-IL-4 toanti-IL-13, there is more anti-IL-13 observed with a slight amount ofanti-IL-4 remaining. Here we see that the ratio of inoculation ismaintained throughout the culture and that the optimization of theprocess could be achieved by balancing the ratios of expressed antibodyhalves through manipulating the started culture ratios.

We have continued to test this process of co-culture expression indelta-Ipp cells on a number of different antibody variants. We show inFIG. 9F the final purified proteins after formulation post HICchromatography of a few exemplary half-antibodies.

Example 4 Heteromultimeric Protein Libraries

This example illustrates the construction of a heteromultimeric proteinlibrary.

Certain methods that may be used to screen mixtures of bispecificantibodies or to rapidly generate large arrays of bispecific antibodiesusing the methods described.

Method #7

In some cases the choice of bispecific antibody is not known, but couldbe the result of the combination of many different half-antibodies.Alternatively, a specific target combination may be desired, e.g.,anti-IL-4/anti-IL-13 but there are a number of candidate half-antibodiesto choose from. Finding the specific half-antibody combination thatyields the best binding or efficacy may be accomplished by combining thehalf-antibodies in a matrix format, one can produce many bispecificantibody variants rapidly. For this experiment, one antibody such asanti-CD3 can be produced at about 10-fold (or greater) excess over theamount of antibody needed for screening. This molecule can then beannealed and oxidized using the procedure described in Example #1. Aboutone tenth of the total amount of the first antibody can be used tocombine with an equal amount of about 10 half-antibodies targetingdifferent antigens (such as anti-CD19, anti-CD20, etc.) as diagramed inFIG. 10. If an additional primary half-antibody is needed to combinewith the second half-antibody repertoire, this can be done to yield aset of screening molecules.

In a second modification of the method, the primary antibody (suchanti-CD3) can be grown as a co-culture using “normal” E. coli host cellsor with a mutant strain having a non-functional lipoprotein phenotype.This half-antibody can then be systematically added to each of thevariable half-antibodies producing an array of bispecific molecules allcontaining the primary targeting half-antibody.

Method #8

The primary half-antibody can be combined with a host of alternativepartnering half-antibodies in a manner that consists of producing thishalf-antibody in sufficient quantity to combine with all of the otherantibody half-antibodies combined. A bulk annealing can then beperformed in a single reaction such that the primary half-antibody iseither the knob or the hole version of the heavy chain and the set ofsecondary targeting half-antibodies are the complimentary mutant. Here,a complex mixture of antibodies can be produced that may be usefultreating disease as a combination.

Alternatively, a co-culture approach using the methods described in theabove Examples can be used to produce a complex mixture of bispecificantibodies with a set primary half-antibody and a variable secondaryhalf-antibody. Such a mixture could then be isolated in bulk and used asa screening material such that a positive result in the pool ofbispecific variants could be later deconvoluted to determine the activebispecific antibody species, or the combined mixture could be used as amore effective therapeutic mixture.

Example 5 In Vitro Activity

This example that the bispecific antibodies described herein possessactivity in in vitro systems. Two cell lines were employed in thisExample 5 and in Example 6, below. In these experiments KP4, apancreatic ductal carcinoma cell line, and A431, an epidermoid carcinomacell line, are both strongly driven by Met or EGFR, respectively,therefore these are good cell lines and tumor xenografts to exploreefficacy of bsAb against each target independently.

The KP4 cell line was obtained from the Riken BioResource Center CellBank (Cell line #: RCB1005, 3-1-1 Koyadai, Tuskuba-shi, Ibaraki 305-0074Japan). The A431 cell line (CRL-1555) was obtained from the AmericanType Culture Collection (ATCC, Manassas, Va.).

Cancer cells, A431, were washed once with PBS, re-suspended inserum-free medium, counted, and then added to 96-well plates (2500cells/well). Cells were then treated with human HGF (0.5 nM) and TGFα□(0.05 nM) alone or with a dose range of either (1) anti-EGFR, (2)Anti-c-met antibody (“one-armed” c-met), (3) the combination ofanti-EGFR and Anti-c-met antibody or (4) the bispecificanti-EGFR/anti-c-met antibody. Three day AlamarBlue™ assays wereperformed according to manufacturer's recommendations (BioSourceInternational; Camarillo, Calif.). IC₅₀ values were determined bynonlinear regression analysis with a four-parameter model (KaleidaGraphver. 3.6, Synergy Software; Reading, Pa.).

In the KP4 cell assay which is Met dependent in vitro and in vivo,growth stimulated by treatment with TGF-alpha and HGF can be inhibitedby Anti-c-met antibody, the combination of Anti-c-met antibody andanti-EGFR, and the bispecific antibody. Treatment with anti-EGFR showslimited activity as a single agent in these cells. There was, however,more potent inhibition by the bispecific antibody in KP4 cells thananti-c-met alone or anti-c-met plus anti-EGFR Abs added separately. InA431 cells, which are primarily driven by EGFR, neither the anti-EGFRantibody nor the anti-c-met antibody alone were able to significantlyinhibit cell proliferation. The combination of both molecules did showsome inhibition of cell proliferation, however, the bispecific antibodyexhibited greater activity at the same concentrations. Also, the cellsexhibited apoptosis in addition to anti-proliferation.

In these assays the bispecific antibody showed improved performancerelative to the other antibodies alone or the combination of anti-Metand anti-EGFR antibodies added separately. These data suggest that it isthe arrangement of anti-Met and anti-EGFR antibodies together on oneantibody that makes the bispecific superior. The results are shown inFIG. 11.

Example 6 In Vivo Activity

This example demonstrates that the bispecific antibodies describedherein possess activity in in vivo models.

Female nude mice that were 6-8 weeks old and weighed 22-30 g wereobtained from Charles River Laboratories, Inc. (Hollister, Calif.). Themice were housed at Genentech in standard rodent micro-isolator cagesand were acclimated to study conditions for at least 3 days before tumorcell implantation. Only animals that appeared to be healthy and thatwere free of obvious abnormalities were used for the study. Allexperimental procedures conformed to the guiding principles of theAmerican Physiology Society and were approved by Genentech'sInstitutional Animal Care and Use Committee. Mice were injectedsubcutaneously with either human KP4 pancreatic cancer cells (5 millioncells in Hank's Balanced Salt Solution (HBSS) plus Matrigel (BDBiosciences) per mouse) or human A431 epidermoid carcinoma cells (5million cells in HBSS plus Matrigel/mouse). When tumors reached ˜150mm³, mice were randomized and treated with vehicle or the bispecificEGFR/c-met (bsEGFR/c-met) (50 mg/kg IP 1×/week) for 2 weeks.

Tumor volumes were measured in two dimensions (length and width) usingUltra Cal-IV calipers (Model 54-10-111; Fred V. Fowler Co.; Newton,Mass.) and analyzed using Excel, version 11.2 (Microsoft Corporation;Redmond Wash.). Tumor inhibition graphs were plotted using KaleidaGraph,version 3.6 (Synergy Software; Reading, Pa.). The tumor volume wascalculated with the following formula:

Tumor size (mm³)=(longer measurement×shorter measurement²)×0.5

The data was analyzed by the mixed modeling approach described below.Here, a strict average and standard deviation are not calculated. Ratherthan provide standard deviations to account for the variability,confidence intervals are used. These are reported in the table as theupper and lower limits in the parenthesis next to AUC/day % TGI. Animalbody weights were measured using an Adventure Pro AV812 scale (OhausCorporation; Pine Brook, N.J.). Graphs were generated usingKaleidaGraph, version 3.6. Percent weight change was calculated usingthe following formula:

Group percent weight change=(new weight−initial weight)/initialweight)×100

To appropriately analyze the repeated measurement of tumor volumes fromthe same animals over time, a mixed modeling approach was used (Pinheiroet al., Linear and Nonlinear Mixed Effects Models. (2008) R packageversion 3.1-89). This approach addresses both repeated measurements andmodest dropouts due to any non-treatment-related deaths of animalsbefore the study end.

Cubic regression splines were used to fit a non-linear profile to thetime courses of loge tumor volume at each dose level. These non-linearprofiles were then related to dose within the mixed model. Tumor growthinhibition as a percentage of vehicle (% TGI) was calculated as thepercentage of the area under the fitted curve (AUC) for the respectivedose group per day in relation to the vehicle, using the followingformula:

% TGI=100×(1−AUC_(dose)/AUC_(veh))

To determine the uncertainty intervals (UIs) for % TGI, the fitted curveand the fitted covariance matrix were used to generate a random sampleas an approximation to the distribution of % TGI. The random sample wascomposed of 1000 simulated realizations of the fitted-mixed model, wherethe % TGI has been recalculated for each realization. The reported UIswere the values for which 95% of the time, the recalculated values of %TGI would fall in this region given the fitted model. The 2.5 and 97.5percentiles of the simulated distribution were used as the upper andlower UIs.

Plotting was performed and generated using R, version 2.8.1 (RDevelopment Core Team 2008; R Foundation for Statistical Computing;Vienna, Austria) and Excel, version 12.0.1 (Microsoft Corporation). Datawere analyzed using R, version 2.8.1, and the mixed models were fitwithin R using the nlme package, version 3.1-89 (Pinheiro et al., 2008).

FIG. 12 & FIG. 13 show the in vivo activity of the anti-EGFR/c-metbispecific antibody in KP4 pancreatic xenograft model and A431epidermoid carcinoma xenograft model, respectively. The bispecificantibody was able to inhibit the growth of the tumors in vivo for bothmodels as compared with control animals that received only the vehicleas a treatment. The graphs indicate the tumor volume was decreased byadministration of the bispecific antibody with the Linear Mixed Effects(LME) fitter tumor volume of 505 mm³ in KP4 xenografts at 20 days aftertreatment compared to 1710 mm³ for the vehicle only arm and 328 mm³ inA431 xenografts after 20 days compared to 495 mm³ in the vehicle onlycontrol. Overall there was a significant change in the AUC/day expressedas % TGI. For the bispecific antibody treatment in the KP4 xenografts,there was 85% tumor growth inhibition (TGI) and in the A431 xenograftmodels there was a 68% TGI.

Example 7 Heteromultimeric Protein Production Using CHO Cell Culture

This example illustrates the formation of heteromultimeric proteins froma culture comprising two host CHO cell populations.

Half-antibodies containing either the knob or hole mutations weregenerated in separate cultures by transiently expressing the heavy andlight chains using constructs and techniques well known in the art.(See, for example, Ye et al., Biotechnol Bioeng. 2009 Jun. 15;103(3):542-51.) Cells were cultured in 1 liter of media (see, forexample, Wong et al., J. Biol. Chem. 2007 282(31):22953-22963) andharvested after 14 days.

Each half antibody was captured on a 5 mL MabSURE SELECT column. Thecolumn was then washed with 10 column volumes (CV) of the equilibrationbuffer followed by 10 CV of a sodium citrate low conductivity buffer(equilibration buffer consisting of 50 mM TRIS pH 8.0, 150 mM NaCl,0.05% Triton X-100, 0.05% Triton X-114; low conductivity wash bufferconsisting of 25 mM Sodium Citrate pH 6.0). Each arm was eluted with0.15 M Sodium Acetate pH 2.7.

Each half antibody was dialyzed into 50 mM TRIS pH 8.0, 150 mM NaCl, 1mM EDTA at a ratio of 1 to 300 at room temperature overnight. Each armwas then centrifuged, filtered using 0.22 micron cellulose acetatefilters and the two arms mixed together at a ratio of 1 to 1 (the totalconcentration was less than 2 mg/mL). The mixture was then processed oneof two ways as follows:

Redox (with water bath): The mixture was then heated in a water bath at37° C. After an hour, the redox mixture was removed from the water bathand left to cool to room temperature. Once the mixture reached roomtemperature, freshly prepared reducing agent, dithiothreitol (DTT), wasadded to achieve a final concentration of 2 mM DTT. The mixture was leftat room temperature for two hours, then concentrated using AmiconUltracell centrifugal filters (10K) to 11 mg/mL and dialyzed into 50 mMTRIS pH 8.0, 150 mL NaCl (1:300) overnight.

Redox (no water bath): The mixture was left at room temperature for 3hours, after which freshly prepared reducing agent, dithiothreitol(DTT), was added to achieve a final concentration of 2 mM DTT. Themixture was left at room temperature for two hours, concentrated usingAmicon Ultracell centrifugal filters (10K) to 11 mg/mL, and dialyzedinto 50 mM TRIS pH 8.0, 150 mL NaCl (1:300) overnight.

Following redox, the assembled material was purified on a 15 mL HICProPac 10 column using a 20 CV gradient similar to the previous section.The running buffer was 25 mM Potassium Phosphate, 0.7M Ammonium SulfatepH 6.5 and the elution buffer was 25 mM Potassium Phosphate pH 6.5, 25%isopropanol. One mL fractions were collected and peak fractions wereseparated by 4-20% Tris-Glycine SDS PAGE to analyze purity and pooledaccordingly. The pools were then concentrated using Amicon Ultracellcentrifugal filters (10K) to around 1.5 mg/mL and dialyzed into 50 mMTRIS pH 8.0, 150 mM NaCl and 0.22 μm filtered.

The identity of each assembled bispecific was confirmed by MassSpectrometry. Purity was analyzed by 4-20% Tris-Glycine SDS PAGE gel andbioanalyzer. Aggregate levels were determined by SEC-MALS.

Results are shown in FIG. 14A, FIG. 14B, and FIG. 140; FIG. 15; FIG.16A, FIG. 16B, FIG. 16C, and FIG. 16D; FIG. 17; FIG. 18; and FIG. 19Aand FIG. 19B. This example demonstrates that bispecific antibodies canbe produced using CHO host cells. One skilled in the art will recognizethat the method can be used to produce other heteromultimeric proteins.

1. A method of preparing a heteromultimeric protein comprising a firsthinge-containing polypeptide having a first heterodimerization domainand a second hinge-containing polypeptide having a secondheterodimerization domain, wherein the second heterodimerization domaininteracts with the first heterodimerization domain, and wherein thefirst and second hinge-containing polypeptides are linked by at leastone interchain disulfide bond, the method comprising the steps of: (a)culturing a first host cell comprising a first nucleic acid encoding thefirst hinge-containing polypeptide under conditions where thehinge-containing polypeptide is expressed; (b) culturing a second hostcell comprising a nucleic acid encoding the second hinge-containingpolypeptide under conditions where the hinge-containing polypeptide isexpressed; (c) disrupting the cell membranes of the first and secondhost cells, wherein the first and second host cells have been combinedtogether in a single suspension; and (d) recovering the heteromultimericprotein, wherein said method does not require the addition of areductant. 2-89. (canceled)